2638 Langmuir 2007, 23, 2638-2646

Synthesis and Aggregation Behavior of Pluronic F87/Poly(acrylic acid)

Block Copolymer in the Presence of Doxorubicin
Yuan Tian, Palaniwasmy Ravi, Lev Bromberg,, T. Alan Hatton,, and Kam C. Tam*,,
Singapore-MIT Alliance and School of Mechanical & Aerospace Engineering, Nanyang Technological
UniVersity, Singapore 639798, and Department of Chemical Engineering, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139

ReceiVed March 23, 2006. In Final Form: NoVember 30, 2006

Poly(acrylic acid) (PAA) was polymerized on both termini of Pluronic F87 copolymer using the atom transfer
radical polymerization technique to produce a novel block copolymer, PAA-b-F87-b-PAA (F87PAA). The loading
of a cationic anticancer drug, doxorubicin (DOX), to F87PAA at different pH values was investigated using isothermal
titration calorimetry (ITC), laser light scattering techniques, and UV-vis spectroscopy. At pH of 4.3-7.1, the ITC
profile exhibited a significant exothermic peak, which indicated that the drug loading is an enthalpically driven process.
At a pH of 4.3, the enthalpy maximum was significantly reduced in the presence of 2 M urea, indicating the existence
of hydrogen bonds between the DOX and F87PAA copolymer. At a pH of 7.1, the fraction of bound DOX was close
to the stoichiometric proportion of 1:1 to the molar concentration of carboxyl groups in the copolymer, where the
drug loading is governed by electrostatic and stacking interactions. The TEM image of the complex indicated the
formation of large compound micelles induced by the binding of DOX to the PAA segments.

Introduction capability and stability of the aqueous Pluronic system, physical

Nano- or microcarriers produced from amphiphilic copolymers
have been explored as potential systems for the delivery of
therapeutic agents over the past decade.1-3 Among them, the
macromolecular surfactants, Pluronic copolymers, have been
studied extensively as a drug delivery vehicle due to their excellent
biocompatibility.4-8 Pluronic block copolymers consist of poly-
(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks
arranged in a triblock PEO-PPO-PEO structure. In an aqueous
environment, Pluronics form micellar structures with a hydro-
phobic PPO core comprises a hydrophobic core, which serves
as a microenvironment for the incorporation of lipophilic drugs.
The corona shell formed by PEO provides steric stabilization,
which enhances the stability and longevity of drug carriers in
blood circulation.9
It was reported that Pluronic copolymers promote active
membrane transport of numerous compounds and thus can help
overcome multiple drug resistance phenomena in cancer
therapy.5-8 Pluronic-based formulations are produced in the form
of hydrogels, water-in-oil, and oil-in-water emulsions, solid
polymer blends, and micelles.5-15 To improve the drug-loading
* Corresponding author. E-mail: mkctam@ntu.edu.sg.
Singapore-MIT Alliance, Nanyang Technological University.
School of Mechanical & Aerospace Engineering, Nanyang Technological
University.
Massachusetts Institute of Technology.
and chemical modification of Pluronic copolymers have been
explored.8,10-14 The physical blending of poly(lactic acid/glycolic
acid) with Pluronic copolymers is a useful approach to produce
stable nanoparticles.10 Such a blend exhibited enhanced stability
in encapsulated proteins and displayed a reduced initial burst
release profile for proteins in their active form.10 The chemical
modification of Pluronic copolymer is an alternative method of
improving its drug-loading capacity. Copolymers of Pluronic
and poly(acrylic acid) (PAA) have been used successfully in
oral delivery of both hydrophobic and hydrophilic anticancer
drugs.12 A one-step synthesis of cationic block copolymers of
poly(2-N-(dimethylaminoethyl) methacrylate) (pDMAEMA) and
Pluronic L92 has been developed for gene delivery.11 The
complexes of the L92-pDMAEMA/DNA have a higher trans-
fection efficacy of the plasmid DNA comparable to that achieved
with lipid-based formulations such as Lipofectamine.
Chemically modified Pluronic copolymers have been devel-
oped for applications in various drug delivery systems.11-13 A
thorough understanding on the interaction between drug and
modified Pluronic copolymer carriers will be important in
optimizing drug delivery, as the controlled drug release correlated
directly with interactions of drugs with its polymeric carriers.
This forms the basic motivation for the current study reported
here. We have selected a PAA-modified Pluronic block copolymer
as the platform and a cationic anticancer drug Doxorubicin (DOX,

(1) Rosler, A.; Vandermeulen, G. W. M.; Klok, H. AdV. Drug DeliVery ReV.
2001, 53, 95.
(2) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf., B 1999, 16, 3.
(3) Torchilin, V. P.; Trubetskoy, V. S. AdV. Drug DeliVery ReV. 1995, 16, 141.
(4) BASF Performance Chemicals, FDA and EPA Status Report. BASF
Corporation: North Mount Olive, NJ, 1993.
(5) Kabanov, A. V.; Batrakova, E. V.; Miller, D. W. AdV. Drug DeliVery ReV.
2003, 55, 151.
(6) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Yu. AdV. Drug DeliVery
ReV. 2002, 54, 759.
(7) Miller, D. W.; Batrakova, E. V.; Alakhov, V. Yu.; Kabanov, A. V.
Bioconjugate Chem. 1997, 8, 649.
(8) Kabanov, A. V.; Lemieux, P.; Vinogradov, S.; Alakhov, V. AdV. Drug
DeliVery ReV. 2002, 54, 223.
(9) Alexandridis, P.; Hatton, T. A. Colloids Surf., A 1995, 96, 1.
(10) Csaba, N.; Gonzalez, L.; Sanchez, A.; Alonso, M. J. J. Biomater. Sci.
Polym. Ed. 2004, 15, 1137.
pKa 8.25)32 as the model drug for examining the interaction
between the drug and modified Pluronic system. In the present
study, PAA chains were grafted to both ends of Pluronic F87,
(11) Bromberg, L.; Deshmukh, S.; Temchenko, M.; Iourtchenko, L.; Alakhov,
V.; Alvarez-Lorenzo, C.; Barreiro-Iglesias, R.; Concheiro, A.; Hatton, T. A.
Bioconjugate Chem. 2005, 16, 626.
(12) (a) Bromberg, L.; Temchenko, M.; Hatton, T. A. Langmuir 2002, 18,
4944. (b) Bromberg, L.; Temchenko, M.; Hatton, T. A. Langmuir 2003, 19, 8675.
(13) (a) Cleary, J.; Bromberg, L.; Magner, E. Langmuir 2004, 20, 9755. (b)
Bromberg, L.; Temchenko, M.; Alakhov, V.; Hatton, T. A. Int. J. Pharm. 2004,
282, 45.
(14) Hoffman, A. S.; Chen, G.; Wu, X.; Ding, Z.; Matsuura, J. E.; Gombots,
W. R. Frontiers in Biomedical Polymer Applications; Ottenbrite, R. M., Ed.;
Technomic Pulishing Co.: Lancaster, PA, 1999; Vol. 2, pp 17-29.
(15) Rapoport, N. Colloids Surf., B 1999, 16, 93.

10.1021/la060780a CCC: $37.00 2007 American Chemical Society

Published on Web 02/02/2007
Pluronic F87/PAA Block Copolymer in Presence of DOX
yielding a novel block copolymer PAA-b-F87-b-PAA (F87PAA).
The reasons for selecting PAA are as follows: (a) it is a pH-
sensitive and biocompatible polymer; (b) at physiological pH,
the negative charges on the PAA can enhance the loading capacity
of oppositely charged species. Pluronic F87 (PEO content of
70%, MW ) 7700, EO ) 2 67 units, PO ) 39 units) was
chosen as a representative example of the Pluronic family because
of its high PEO ratio and narrow polydispersity (PDI ) 1.15).
PEO provides a steric barrier against self-aggregation and
unfavorable interactions with albumin or cellular components in
the bloodstream. The drug-loading procedures and drug-polymer
interaction were studied using isothermal titration calorimetric
(ITC), dynamic light scattering (DLS), and UV-vis spectroscopic
techniques. The formation of polyion complexes (PIC) with the
oppositely charged anticancer drug Doxorubicin (DOX) in
aqueous solutions was elucidated.
Experimental Section
Materials. Pluronic F87 was obtained from BASF Corporation
(Mount Olive, NJ). Trace amounts of water in F87 were removed
using azeotropic distillation prior to use. tert-Butyl acrylate (tBA)
(Aldrich, 99%) was passed through a basic alumina column, dried
over CaH2, and vacuum-distilled before polymerization. Triethyl-
amine (TEA) and toluene were distilled before use. CuBr (99.99%),
N,N,N,N,N-pentamiethyldiethylenetriamine (PMDETA), 2-bro- with 1 M standard NaOH solution was performed at 25 C under
moisobutyl bromide, tetrahydrofuran (THF), hexane, and methanol
were purchased from Aldrich and used as received. Doxorubicin
hydrochloride (99%) was purchased from Hande Tech USA (Houston,
TX) and used without further purification.
Synthesis of Bromo-Terminated F87 Macroinitiator (Br-F87-
Br). All the synthetic steps were carried out under an argon
atmosphere. In a three-neck round-bottom flask, F87 (10 g, 1.3
mmol) was dissolved in freshly distilled toluene (180 mL) at room
temperature. The trace amount of water in F87 was removed using
azeotropic distillation. The mixture was cooled to 0 C, deoxygenated
triethylamine (0.543 mL, 3.89 mmol) was added with stirring, and
2-bromoisobutryl bromide (0.482 mL, 3.9 mmol) in dry toluene
(10 mL) was added dropwise at 0 C over a 1 h period through
a pressure-equalizer funnel under an argon atmosphere. After 24 h
of reaction at room temperature, the resulting white solid was filtered
and the toluene removed by rotary evaporation. Macroinitiator, Br-
F87-Br, was precipitated in excess n-hexane and dried under vacuum.
The crude macroinitiator was dissolved in water (pH 8) and
extracted three times using dichloromethane. The organic layers
were collected and dried over MgSO4, and the final product was
recovered by precipitation in n-hexane and dried overnight under
vacuum at room temperature. Yield: 6 g (60%). MnGPC ) 9600 Da,
Mw/Mn ) 1.13.
Preparation of F87PAA Block Copolymer. Difunctional bromo-
terminated F87 macroinitiator (0.5 g, 0.125 mmol of Br) and CuBr
(0.018 g, 0.125 mmol) were added to a predried Schlenk flask,
which was then sealed with a rubber septum. Deoxygenated toluene
and tBA (2.4 mL, 16.5 mmol) were added via a syringe that had
been purged with argon prior to use. The mixture was stirred until
the Br-F87-Br macroinitiator was totally dissolved and then evacuated
with three freeze-thaw cycles to remove oxygen. The degassed
PMDETA (26 L, 0.125 mmol) was added, and the solution was
stirred until the Cu/ligand complex had formed. This was easily
visualized through a change in the solution from a white cloudy to
a clear, light green solution. The reaction mixture was stirred at 80
C for 6 h. After completion of the polymerization, the mixture was
quenched by exposing it to air and eluted through a neutral alumina
and ion-exchange resin to remove the catalyst. The solution was
concentrated and precipitated into an excess of water:MeOH 50:50
(v/v) solution. The precipitation procedure was repeated twice and
the polymer was dried under vacuum at room temperature for 3 days
to obtain the PtBA-b-F87-b-PtBA (F87PtBA) copolymer. Yield:
65%. MnGPC ) 31800; Mw/Mn ) 1.15.
Langmuir, Vol. 23, No. 5, 2007 2639
The molar composition of the F87PtBA copolymer was determined
from the 1H NMR spectrum using the relative peak intensity at 1.43
ppm (-C(CH3)3 of the tBA block), and 1.13 ppm methyl protons
of PPO block. Based on the 1H NMR and GPC results, the degree
of polymerization was determined and found to be 170 tBA units.
Subsequently, hydrolysis was performed by adding an excess of
trifluoroacetic acid (TFA) to the copolymer solution in methylene
chloride and stirred at room temperature according to the procedure
reported in the literature.16-18 After 24 h of reaction time, the solvent
was concentrated and precipitated in excess hexane, leading to the
targeted F87PAA block copolymer. The absorbance bands in the
1650-1700 and 1100 cm-1 regions, characteristic for COOH in
PAA and the C-O-C stretch in Pluronic, were observed by FTIR
(KBr pellet).19 The content of the carboxylic group was quantified
by potentiometric titration.
Polymer Characterization. GPC of F87 macroinitiator and block
copolymer F87PtBA was performed on an Agilent 1100 apparatus
(Germany) equipped with a liquid chromatography pump, photo-
luminescence gel (5 mm MIXED-C column), and differential
refractometer as the detector. The column was calibrated with narrow
molecular weight polystyrene standards. THF was used as the mobile
phase, at a flow rate of 1.0 mL/min. 1H NMR spectra were recorded
at room temperature using a Bruker ACF-400 (400 MHz) spec-
trometer and the chemical shifts () were given in ppm using
tetramethylsilane (TMS) as an internal reference.
Potentiometric Titration. To obtain the ion-exchange capacity
of F87PAA block copolymer, titration of 0.1 wt % polymer solution
constant stirring. An ABU93 Triburet Titration system equipped
with Radiometer pHG201 pH glass and Radiometer REF201
reference electrodes was used for pH measurement. The degree of
neutralization, RN, of carboxylic groups is defined by
[BASE] + [H+] - [OH-]
RN ) (1)
C[COOH]
Here, [BASE], [H+], and [OH-] are the molar concentrations of
added NaOH, free hydrogen ion, and hydroxide ion, respectively,
and C[COOH] is the total concentration of carboxylic groups.
Dynamic Light Scattering (DLS). Doxorubicin solution (15 mM)
was prepared in deionized water and stored at 4 C prior to use.
Stock solutions of F87PAA were prepared in deionized water in an
ice bath. The DOX solution (15 mM) was injected into 0.01 wt %
polymer solution at 25 C using a microsyringe. Then the mixture
was kept in the water bath for 12 h under continuous shaking at 25
C. DLS measurements were carried out using a Brookhaven ZetaPlus
Analyzer. The particle sizes were measured at room temperature
using DLS at different DOX concentrations at a scattering angle of
90. A solid-state 671 nm laser was used as the light source. The
time correlation function of the scattered intensity G2(t) ) I(t)
I(t + t) was analyzed using the inverse Laplace transformation
technique with the GENDIST software package to analyze the
distribution function of the decay times. The apparent hydrodynamic
radius, Rhapp, was determined using the Stokes-Einstein equation,
kT
Rh ) (2)
6D
where D is the translational diffusion coefficient, which was calculated
from the decay rate ) Dq2, k is the Boltzmann constant, is the
solvent viscosity, q is the scattering vector (q ) (4n sin(/2))/),
n is the refractive index of the solution, is the scattering angle,
and is the wavelength of the incident laser light in a vacuum.
Isothermal Titration Calorimetry. Calorimetric experiments
were performed using a Microcal Isothermal Titration Calorimeter
(16) Ma, Q.; Wooley, K. L. J. Polym. Sci., Part A: Polym. Chem. 2002, 38,
4805.
(17) Hou, S. J.; Chaikof, E. L.; Taton, D.; Gnanou, Y. Macromolecules 2003,
36, 3874.
(18) Lu, Z. H.; Liu, G. J. Macromolecules 2004, 37, 174.
(19) Bromberg, L. J. Phys. Chem. B 1998, 102, 1956.
2640 Langmuir, Vol. 23, No. 5, 2007
Scheme 1. Synthesis of F87PAA Block Copolymer by ATRP
(ITC) (MicroCal, Northampton, MA). A sample cell with a volume
of 1.35 mL was filled with a 0.01 wt % polymer solution. For precise
measurements, each injection should result in an average of at least
5-10 cal of heat absorbed or evolved into the 1.35 mL cell.20 Our
titration experiments were carried out at 25 C by injecting 15 mM
DOX solutions from a 250 L injection syringe into the sample cell,
which was stirred at 400 rpm to ensure an optimum mixing efficiency,
where the heat evolved is about 15 cal. In selecting the optimum
stirring rate, there is a trade-off between mixing efficiency and
baseline noise. For all studies, stirring rates of ca. 400-500 rpm
gave adequate mixing following each injection, while still providing
very high baseline quality.20 The observed released heat data included
the heat of dilution for the DOX in water and heat of DOX binding
to the polymer chains. The heat of dilution can be determined by
performing a blank titration in aqueous solution without polymer.
In this study, the differential enthalpy curves of DOX binding to
F87PAA were subtracted from heat of dilution of DOX. Data analysis
was performed using Microcal ORIGIN software.
The samples from the ITC experiments were centrifuged at 8000
rpm (30 min). The uptake of doxorubicin by the polymer was assayed
using UV-vis spectrophotometry. Free DOX in supernatant solutions
of the drug/polymer suspensions was measured at ) 481 nm,
using an extinction coefficient,  ) 10410 M-1 cm-1 at 25 C.
Transmission Electron Microscope (TEM). TEM was performed
on a JEOL JEM-2010 electron microscope at an acceleration voltage
of 120 kV. The sample was prepared on a 400 mesh copper grid
precoated with a carbon film and stained with phosphotungstic acid
(0.1 wt %, ethanol).
Results and Discussion
Polymer Synthesis. PAA-b-F87-b-PAA (F87PAA) was
synthesized by the ATRP technique using protected group
chemistry, followed by hydrolysis in acidic conditions. The
(20) MicroCal Inc. Micro Calorimetry System, Users Manual; MicroCal, LLC:
Northampton, MA, 1993; Version 2.9, p 75.
Tian et al.
copolymerization of tert-butyl acrylate (tBA) by the atom transfer
radical polymerization (ATRP) technique has been reported with
polystyrene, PEO, PCL, or other macroinitiators.16-18 Scheme
1 shows the general procedure for the synthesis of F87PAA from
F87. Under copper-mediated ATRP conditions, multidentate
N-containing ligands have been used in the polymerization
process.21 Among these ligands, polymerization in the presence
of multidentate alkyl amino ligands, such as pentamethyldieth-
ylenetriamine (PMDETA), has been found to proceed at a faster
rate and lower temperature.22 In this study, the block copolymer
F87PtBA was synthesized by ATRP using the bromide-terminated
Pluronic F87 macroinitiator (Br-F87-Br) and a CuBr/PMEDETA
complex as the catalyst in toluene at 80 C. The molar ratio of
the macroinitiator:CuBr:PMEDETA employed was 1:2:2. The
GPC trace of (Figure 1) PtBA-b-F87 showed a monomodal
distribution peak with Mn of 31800 Da and narrow molecular
weight distribution (PDI ) 1.15). The absence of tailing or
multiple peaks indicated the successful polymerization of tBA
with F87. Subsequently, the protecting tBA groups were
hydrolyzed in the presence of TFA in dichloromethane to obtain
F87PAA.
Isothermal Titration Calorimetry (ITC). To further inves-
tigate the binding of DOX with F87PAA block copolymer,
calorimetric experiments were employed to study the enthalpy
changes associated with interaction between the drug and polymer.
The differential enthalpy curves for titrating 15 mM DOX into
0.01 wt % F87PAA solution are shown in Figure 2. Over the pH
range of 4.3 to 7.1, the enthalpy profile corresponding to the
DOX/F87PAA binding process possessed an exothermic peak.
The onset of the exothermic peak, namely, C1, was 0.02 mM.
The exothermic peak became broader with an increase in the pH,
(21) Matyjaszewski, K.; Xia, J. Chem. ReV. 2001, 101, 2921.
(22) Xia, J.; Matyjaszewski, K. Macromolecules 1997, 30, 7697.
Pluronic F87/PAA Block Copolymer in Presence of DOX
Figure 1. GPC trace of F87 macroinitiator and F87PtBA block
copolymer.
Figure 2. Differential enthalpy curves of titrating 15 mM doxorubicin
into 0.01 wt % F87PAA solutions at different pH at 25 C.
which suggested that more DOX molecules were bound to the
polymer chains as the pH was increased. This resulted in a higher
drug binding at the equilibrium point, designated as C2. The
binding fraction of DOX in the polymer chain is given by the
following expression,
C 2 - C1
) (3)
C[COOH]
where C1 and C2 were determined from the differential enthalpy
curves (Figure 2). C[COOH] (0.76 mM) is the maximum ion-
exchange capacity of COOH groups determined from poten-
tiometric titration. The values of C1, C2, binding fraction , and
neutralization degree (RN) of F87PAA at different pHs are
summarized in Table 1.
At pH 2.5, F87PAA block copolymer is neutral due to the
protonation of PAA segments, and the inter- and/or intrapolymer
complex formed through hydrogen bonding between protons of
carboxylic acid and ether oxygens of F87 block, i.e.,
or between carboxylic groups, i.e.,
The absence of any appreciable change (Figure 2) at this pH
indicated only a very weak interaction between DOX and
Langmuir, Vol. 23, No. 5, 2007 2641
Table 1. Binding Fraction of DOX (15 mM) in 0.01 wt %
F87PAA Solution at Different pH
pH RN C1 (mM) C2 (mM)
2.5 0 NA NA NA
4.3 0.05 0.02 0.32 0.41
4.9 0.1 0.02 0.34 0.43
5.4 0.2 0.02 0.35 0.45
6.0 0.4 0.02 0.43 0.55
6.6 0.6 0.02 0.60 0.78
7.1 0.8 0.02 0.65 0.85
unionized F87PAA. The small exothermic peak may be related
to weak hydrophobic interactions between the PPO segments of
F87PAA and the hydrophobic anthraquinone segment on the
DOX molecule. At a pH of 4.3, we expected the interaction
between F87PAA and DOX to be weak because the neutralization
degree of carboxylic groups on the PAA chains was only 5% as
determined from potentiometric titration. However, the distinct
exothermic peak evident in the ITC binding curves (Figure 2)
indicated that DOX interacted strongly with F87PAA even in
the presence of a small amount of negative charge on the PAA
segments. But the absence of any appreciable enthalpy change
at pH 2.5 indicated only a very weak interaction between DOX
and unionized F87PAA. This suggested that the electrostatic
force is a prerequisite for the onset of the interaction between
F87PAA and DOX molecules. We noted that the binding fraction
() of DOX (Table 1) at low pH (pH of 4.3-5.3) was almost
independent of the neutralization degree of the polymer, which
suggested that forces other than electrostatic interaction may be
involved in the binding of DOX to the polymer chains. It is
instructive at this point to consider the complexation of DOX/
DNA. The substituent (HO-C-CO-CH2OH) near the an-
thraquinone ring of DOX defines the functional domain. This
substituent formed hydrogen bonds with DNA bases, serving as
an anchor to stabilize the complex.23 It has been reported24,25 that
hydrogen bonding (H-bonding) between anionic polymers (e.g.,
poly(acrylic acid) or polyaspartic acid) and cationic drugs (e.g.,
Procaine HCl or diminazene) can contribute to the stability of
drug-polymer complexes. Therefore, we hypothesized that
H-bonding between DOX and -COOH of F87PAA, as well as
the long-range electrostatic interaction, may play an important
role in the formation of the F87PAA/DOX complex. To verify
this hypothesis, the effect of urea on the DOX/polymer interaction
was studied using ITC. Urea is a well-known chaotropic agent26
that can disrupt H-bonding by forming H-bonds with both proton
donors and acceptors. The enthalpy curves in Figure 3 showed
the effect of the absence or presence of 2 M urea on the observed
enthalpy during the titration of 10 mM DOX solution into a
F87PAA solution at pH 4.3. The maximum enthalpy was reduced
significantly from -21.9 to -13.6 kJ/mol upon the addition of
2 M urea, which agreed with the value of enthalpy contribution
from the hydrogen bonding (-5 to -20 kJ/mol).26 This indicated
that H-bonding between F87PAA and DOX was weakened since
most of the proton-donating and -accepting sites on both molecules
were occupied by urea molecules.
Additionally, the pH decreased upon the addition of DOX
to a F87PAA solution in the low pH region, as shown in Figure
4a, where a considerable pH reduction, from 4.3 to 3.7, was
observed. Above CDOX ) 0.3 mM no further reduction inpH
(23) Chaires, J. B.; Satyanarayana, S.; Suh, D.; Fokt, I.; Przewloka, T.; Priebe,
W. Biochemistry 1996, 35, 2047.
(24) Ehtezazi, T.; Govender, T.; Stolnik, S. Pharm. Res. 2000, 17, 871.
(25) Govender, T.; Ehtezazi, T.; Stolnik, S.; Illum, L.; Davis, S. S. Pharm. Res.
1999, 17, 1125.
(26) Mathews, C. K.; van Holde, K. E. Biochemistry; The Benjamin/Cummings
Publishing Co.: Redwood City, CA, 1990.
2642 Langmuir, Vol. 23, No. 5, 2007
Figure 3. Enthalpy curves for titrating 10 mM DOX into 0.0067
wt % F87PAA at pH 4.3: (9) in the absence of urea; (0) in the
presence of 2 M urea.
Figure 4. Drug-loading profiles obtained from titrating 15 mM
DOX into 0.01 wt % F87PAA solution at pH 4.3, 25 C: (a) (9)
pH measurement; ([) turbidity at ) 600 nm. (b) (2) Differential
enthalpy curve of titrating 15 mM doxorubicin into 0.01 wt %
F87PAA solutions at pH 4.3.
occurred. The degree of ionization R is derived from the
equation,
[H+] - [OH-]
R ) RN + (4)
C[COOH]
where [H+] and [OH-] were calculated from the measured pH.
Applying eq 4 to F87PAA (RN ) 0.05) at C[COOH] ) 0.76 mM,
we determined the degree of ionization to be R ) 0.3 at pH of
3.7, indicating that nearly 25% of COOH groups was ionized
following the addition of DOX. Turbidimetric titration (Figure
4a) showed that the absorbance ( ) 600 nm) of the solution
increased gradually upon the addition of DOX up to a
concentration of 0.3 mM, which indicated the formation of
polymer/drug complexes. This process occurred with a significant
enthalpy change, which also ceased at a similar drug loading
concentration equilibrium point (Figure 4b). The blank titration
was performed at the same pH to ensure that the reduction in
the pH was not due to the dilution of DOX in water. Hence, we
believe that the addition of DOX caused the deprotonation of
Tian et al.
Figure 5. Schematic representation of DOX loading procedure at
pH 4.3.
carboxylic groups on PAA segments, which was supported by
the findings of Kogej,27 where the degree of dissociation in
solution of unneutralized poly(acrylic acid) increased upon the
addition of oppositely charged surfactants. The increase in the
ionization degree of PAA segments with the addition of DOX
may transform the environment of PPO segments to one that is
more polar, which may induce the PPO segments to aggregate.28
Figure 5 shows a schematic representation of the drug-loading
process at pH 4.3. Initially (stage I), carboxylic groups on F87PAA
chains were slightly dissociated into R[COO-]m and H+ to
maintain a dissociation equilibrium between these species at pH
4.3. When DOX solution was added to the polymer solution
(stage II), positively charged DOX molecules partitioned to the
polymer chains and interacted with R[COO-]m segments, leading
to the formation of F87PAA/DOX complexes in stage II. The
dissociation equilibrium of carboxylic groups in stage I was
disrupted due to the reduction in the concentration of R[COO-]m.
In stage III, further dissociation of R[COOH]n resulted in a new
(27) Kogej, K. J. Phys. Chem. B 2003, 107, 8003.
(28) Bromberg, L. J. Phys. Chem. B 1998, 102, 10736.
Pluronic F87/PAA Block Copolymer in Presence of DOX Langmuir, Vol. 23, No. 5, 2007 2643

Figure 7. UV measurements of DOX loading at pH 4.3-7.1.

Figure 6. (a) UV-vis absorbance of DOX and polymer-bound

DOX; (b) fluorescence emission of DOX and polymer-bound DOX
at pH 7.1.

equilibrium via the deprotonation of carboxylic groups and the

release of protons to the bulk solution, as reflected by the pH
reduction during the drug-loading process.
UV Absorption and Fluorescence Emission Measurements
on the Complexation of DOX with F87PAA Block Copolymer.
Figure 6a shows the UV-vis absorbance spectra of free and
polymer-bound DOX in a pH 7 buffer. Binding of the drug to
F87PAA copolymer resulted in a 10-nm red shift of the maximum
absorbance at 480 nm. The red shift resulted from stacking
interactions of DOX molecules immobilized along PAA chains.29
The tendency of the aromatic chromophore of DOX to self- Figure 8. Relaxation time distribution functions of 0.01 wt %
F87PAA solution at different DOX concentrations at 25 C and
association is well-documented.30,34 The chromophore of DOX
scattering angle 90.
exhibited unique fluorescence characteristics that are sensitive
to changes in the microenvironment of the drug.30,31 The
fluorescence spectrum of DOX consisted of a broad emission DOX complex formation is strongly dependent on the pH as the
band with a maximum at 590 nm and a shoulder at 558 nm, as degree of carboxyl group ionization increased with pH, and the
shown in Figure 6b. The addition of F87PAA to the DOX solution uptake of DOX agreed with the binding fraction of DOX (Table
resulted in an overall reduction in the fluorescence intensity of 1) as determined from ITC studies. This result indicated that the
the drug, which was not accompanied by a shift in the wavelength electrostatic interaction played a major role in the drug loading
of emission. This reduced fluorescence intensity of DOX in the in the F87PAA system. Similar results were observed with poly-
presence of F87PAA is related to a self-quenching of bound (acrylic acid)32 and polyether-modified poly(acrylic acid) mi-
DOX as the polyacylate anion is not a fluorescence quencher.30,32 crogels.12 Binding of DOX to poly(acrylic acid) resulted in a
The equilibrium uptake of DOX to F87PAA, determined by maximum drug loading at pH 6.5, accompanied by a deteriorating
UV measurement, is shown in Figure 7, where the DOX binding DOX binding with a decrease with the reduction in the ionization
increased with increasing pH. At a pH of 6.1-7.1, F87PAA/ of DOX at high pH.32
Dynamic Light Scattering (DLS). Dynamic light scattering
(29) Kitaeva, M. V.; Melik-Nubarov, N. S.; Menger, F. M.; Yaroslavov, was carried out to study the formation of DOX/F87PAA complex
A. A. Langmuir 2004, 20, 6575.
(30) Husain, N.; Agharia, R. A.; Warner, I. M. J. Phys. Chem. 1993, 97, at a pH of 7.1. The relaxation time distribution functions of
10857. F87PAA solutions at different DOX concentrations are shown
(31) Li, X.; Hirsh, D. J.; Cabral-Lilly, D.; Zirkel, A.; Gruner, S. M.; Janoff,
A. S.; Perkins, W. R. Biochim. Biophys. Acta 1998, 1415, 23.
in Figure 8. Before the addition of DOX, the scattering intensity
(32) Kitaeva, M. V.; Melik-Nubarov, N. S.; Menger, F. M.; Yaroslavov, was extremely low and the g2(t) correlation function cannot be
A. A. Langmuir 2004, 20, 6575. accurately determined, suggesting the presence of only unimeric
(33) Mayer, L. D.; Bally, M. B.; Cullis, P. R. Biochim. Biophys. Acta 1986,
857, 123-126. polymer chains. When DOX was added, the presence of a narrow
(34) Aubel-Sadron, G.; Londos-Gagliardi, D. Biochimie 1984, 66, 333-352. peak indicated the formation of the polymer/drug complex. The
2644 Langmuir, Vol. 23, No. 5, 2007 Tian et al.

Figure 9. Drug-binding profiles obtained from titration of 15 mM

DOX into 0.01 wt % F87PAA solution at pH ) 7.1 (R ) 0.8),
25 C: (a) Absorbance measurement at ) 600 nm; (b) dependence
of Rhapp on DOX concentration.

solution remained optically transparent, with no appreciable

absorbance being observed during the turbidity measurements
(Figure 9a). Figure 9b shows the apparent hydrodynamic radius
of the polymer/DOX complex, Rhapp, plotted against DOX
concentration. In region I (0.024 mM e [DOX] e 0.13 mM),
Rhapp of the polymer/drug complex was found to be in the range
of 110-150 nm. In contrast to the PAA homopolymer (Mn )
5000 Da) system, which formed large (600-900 nm) particles
with DOX ([DOX] ) 50 M),32 the F87PAA/DOX complexes
may be stabilized by the steric effect of PEO segments of the
Pluronics. At [DOX] > 0.13 mM (region II), two diffusive modes
were observed in Figure 8, where the slow mode corresponded
to large aggregates with particles sizes 440 nm (see Figure 9b).
The turbidity measurements in Figure 9a showed a gradual
increase in the turbidity as the solution became slightly opaque.
Figure 10. (a) TEM image of F87PAA/DOX complex (sample
The neutralization of the polymer by bound drug in the complex prepared at pH 7, [DOX]/[COOH] ) 0.07); (b) proposed mechanism
contributed to the formation of large colloidally unstable polymer/ for DOX binding to F87PAA.
DOX aggregates. Beyond [DOX] ) 0.34 mM, the binding
approached region III, characterized by a sharp increase in the molecules, electrostatic repulsion decreased due to increasing
absorbance, consistent with the formation of large aggregates, number of [COO-] sites being neutralized by drug molecules,
which ultimately resulted in phase separation. A large fraction as well as the enhanced hydrophobicity of the DOX/F87PAA
of the hydrophobic domain (anthraquinone in DOX) was exposed complex since more anthraquinone rings were present within the
to the water phase and PEO segments cannot stabilize the complex. The reduced electrostatic repulsion and increased
F87PAA/DOX complexes at these high DOX loadings. hydrophobicity of the DOX/F87PAA complex led to the formation
On the basis of the results obtained from DLS and turbidity of the larger aggregates observed at higher DOX concentration.
measurements, the observed aggregation of DOX/F87PAA was Transmission Electron Microscopy (TEM). Transmission
a concentration-dependent process. DOX molecule possessed a electron microscopic studies were also carried out to investigate
planar hydrophobic aromatic anthraquinone ring, and protonated the microstructure of F87PAA/DOX complex. The TEM image
amino nitrogen with a positive charge.33 DOX binding with the of the sample prepared at pH of 7 is shown in Figure 10a for
F87PAA block copolymer can be represented by [DOX]/[COOH] ) 0.07. The sizes of the complexes were about
200 nm, which is consistent with the particle size obtained from
COO- + Na+ + DOX+ + Cl- S light scattering measurements. The self-association of polymer-
COO-DOX+ + Na+ + Cl- bound DOX together with free polymeric chains produced
physical cross-linking that resulted in the formation of F87PAA/
where the DOX/F87PAA complex is stabilized by the electrostatic DOX complex as shown in Figure 10a. The proposed mechanism
repulsion of [COO-] from PAA segments and steric effect of of the complexation induced by DOX binding is illustrated in
PEO block from F87 segment. Upon the binding of more DOX Figure 10b.
Pluronic F87/PAA Block Copolymer in Presence of DOX
Figure 11. (a) Transmittance at 600 nm and light scattering intensity
at 671 nm. Sample was prepared from 0.1 wt % F87PAA at 25 C;
(b) dependence of decay rate on q2 for 0.085 wt % F87PAA block
copolymer solution at pH 3.5.
Figure 12. (a) Drug-binding profiles obtained from titrating of 15
mM DOX into 0.01 wt % F87PAA solution at pH ) 3.5. Dependence
of Rhapp on DOX binding; (b) dependence of scattering intensity on
DOX binding at pH 3.5. Scattering angle of 90.
F87PAA and F87PAA/DOX at Very Low pH. The existence
of H-bonded complexes within F87PAA copolymer at pH 3.5
was demonstrated by examining the transmittance and light
scattering intensity (Figure 11a). At this pH, the PAA segments
are not ionized since the pK0 of F87PAA is about 4.8. At pH
3.5, the transmittance from UV measurement was significantly
reduced and the scattering intensity from DLS was large as
Langmuir, Vol. 23, No. 5, 2007 2645
Figure 13. Morphology of F87PAA particles: (a) [DOX] ) 0 mM;
(b) [DOX] ) 0.05 mM ([DOX]/[COOH] ) 0.07). Sample directly
prepared from DLS experiments.
observed from DLS experiment, suggesting the existence of inter-
or intramolecular complex.
To investigate the formation of H-bonded inter- or intramo-
lecular complexes, the size of 0.085 wt % F87PAA (pH 3.5)
complex in the presence and absence of 0.22 M ethylene glycol
was examined. The distribution functions of F87PAA in ethylene
glycol-water and water were obtained at varying scattering
angles. The dependence of decay rate exhibited a linear
relationship with the square of the scattering vector q2 (Figure
11b), indicating that the distribution function was due to the
translational diffusion of the complex. In the absence of ethylene
glycol, the average hydrodynamic radius of F87PAA complex
was 126.4 nm, and it decreased to 71.2 nm in the presence of
0.22 M ethylene glycol. The ethylene glycol is a strong hydrogen-
bonding agent, which can disrupt H-bonding by forming H-bonds
with both proton donors and acceptors. The reduction in the size
of the complex with the addition of ethylene glycol suggested
that inter- or intramolecular H-bonded complexes were present
in F87PAA block copolymer at pH 3.5.
DLS was carried out to investigate the dissociation behavior
of F87PAA block polymer in aqueous solution, and we found
that soluble complexes with a detectable size were only observed
within a narrow pH window (between pH 3.4 and pH 3.9). For
0.01 wt % F87PAA solution (note: this concentration was used
in the DOX binding studies at pH 7.1 as discussed earlier) at pH
3.5, Rh of the F87PAA complex was about 70 nm. When DOX
was added, the presence of a narrow peak and enhanced light
scattering intensity indicated the formation of polymer/drug
complex. This binding may be initiated by hydrogen bonding
between DOX and unionized PAA or PEO segments. The effect
of DOX binding on the size of the complex at pH 3.5 is shown
in Figure 12. The scattering intensity of F87PAA solution was
2646 Langmuir, Vol. 23, No. 5, 2007
only about 10 kcps in the absence of DOXand increased to 34
kcps in the presence of 0.02 mM DOX ([DOX]/[COOH] ) 0.03,
molar ratio), as shown in Figure 12b. The scattering intensity
reached a plateau (530 kcps) with the addition of 0.075 mM
DOX. The particle size of F87PAA in the absence of DOX was
70 nm, increased to 110 nm with the addition of DOX, and
remained constant when the drug concentration was lower than
0.075 mM ([DOX]/[COOH] ) 0.10), beyond which the formation
of large aggregates (Rh > 500 nm) was observed (Figure 12a).
Figure 13 shows the TEM micrographs depicting the morphology
of F87PAA complex in the presence and absence of DOX. As
shown in Figure 13a, spherical F87PAA complex was produced
at pH 3.5, where inter- or intramolecular hydrogen bonding took
place between the carboxylic groups on the PAA segment and
PEO block, leading to the formation of soluble complexes. With
the addition of drug molecules, large and compact complexes
were observed (Figure 13b) indicating the formation of F87PAA/
DOX complexes induced by DOX binding. The diameter of the
complexes is about 200 nm, which is consistent with that
determined from dynamic light scattering.
Conclusions
A block copolymer Pluronic-poly(acrylic acid) (F87PAA)
was synthesized using the atom transfer radical polymerization
technique. Over a wide pH range, the ITC profile exhibited a
significant exothermic peak, which indicated that the drug loading
is an enthalpically driven process. Long-range electrostatic
interactions dominated the interaction between F87PAA and DOX
molecules. At a pH of 4.3 where the F87PAA possessed a low
neutralization degree, the drug loading was induced by small
Tian et al.
amounts of COO-, followed by the deprotonation of uncharged
carboxylic groups via the poly(acrylic acid) dissociation equi-
librium. Polymer/drug complexes were stabilized by hydrogen
bonding. At pH of 7.1 (R ) 0.8), the aggregation behavior of
F87PAA block copolymer is strongly dependent on the drug
concentration, where the drug loading is controlled by electrostatic
and stacking interactions. TEM micrograph showed that large
spherical F87PAA/DOX complexes were formed by DOX
binding.
The electrostatic interaction as well as stacking interaction
between ionized PAA segments and DOX is believed to be the
basis for the drug binding with the PAA-modified Pluronic that
stabilized the DOX/F87PAA complex. At pH 7.2, the equilibrium
uptake of DOX is close to the stiochiometric 1:1 ratio of DOX
and concentration of carboxyl groups, indicating the high binding
affinity of DOX within the complex at pH 7.2. At pH 5.0, DOX
content in the complex reduced to 60% of the overall ion-exchange
capacity due to the lower ionization degree that decreased with
decreasing pH. We believe that the protonation of carboxyl groups
reduced the uptake of DOX by F87PAA block copolymer, which
can be utilized for the pH-induced release of DOX in physiological
pH range (pH 7.4-5.0). Studies of stability of the complexes
and DOX release from DOX/F87PAA complex in response to
pH changes are currently in progress.
Acknowledgment. We thank Drs. Wang Chang, Dai Sheng,
and Xiong Xiang Yuan for their helpful suggestions. We
acknowledge the financial support provided by the Singapore-
MIT Alliance.
LA060780A