Biomacromolecules 2004, 5, 463-473

463

Controllable Surface Modification of Poly(lactic-co-glycolic

acid) (PLGA) by Hydrolysis or Aminolysis I: Physical,
Chemical, and Theoretical Aspects
Tristan I. Croll, Andrea J. OConnor, Geoffrey W. Stevens, and Justin J. Cooper-White*
Department of Chemical and Biomolecular Engineering, The University of Melbourne,
Victoria 3010 Australia
Received August 18, 2003; Revised Manuscript Received October 30, 2003

While biodegradable, biocompatible polyesters such as poly (lactic-co-glycolic acid) (PLGA) are popular
materials for the manufacture of tissue engineering scaffolds, their surface properties are not particularly
suitable for directed tissue growth. Although a number of approaches to chemically modify the PLGA surface
have been reported, their applicability to soft tissue scaffolds, which combine large volumes, complex shapes,
and extremely fine structures, is questionable. In this paper, we describe two wet-chemical methods, base
hydrolysis and aminolysis, to introduce useful levels of carboxylic acid or primary and secondary amine
groups, respectively, onto the surface of PLGA with minimal degradation. The effects of temperature,
concentration, pH, and solvent type on the kinetics of these reactions are studied by following changes in
the wettability of the PLGA using contact angle measurements. In addition, the treated surfaces are studied
using X-ray photoelectron spectroscopy (XPS) to determine the effect on the surface chemical structure.
Furthermore, we show using XPS analysis that these carboxyl and amine groups are readily activated to
allow the covalent attachment of biological macromolecules.
Introduction
Soft tissue engineering offers a number of unique challenges not seen in other tissue engineering applications such
as bone, cartilage or skin. For many soft tissue applications,
scaffolds must be large (e.g. breast reconstruction1), very
highly porous, and soft, yet have enough strength to resist
the contractile forces generated by growing tissue. Scaffolds
produced from poly(lactic-co-glycolic acid) (PLGA) using
thermally induced phase separation (TIPS)2,3 are able to meet
these goals. However, it is well-known that the surface
properties of PLGA are not ideal for cell growth.4-7 PLGA
is relatively hydrophobic compared to the natural extracellular matrix (ECM), is unable to interact specifically with
cells, and does not possess any functional groups for the
attachment of biologically active molecules.
Since the mechanical and degradative properties of PLGA
are seemingly ideal, and given that PLGA is approved by
the U.S. Food and Drug Administration (FDA) and other
regulatory bodies for implantation into humans, a promising
approach appears to be the surface modification of PLGA
scaffolds post-formation. Ideally, this gives useful surface
characteristics to the polymer, without changing the properties of the bulk.
Many approaches have been taken to modify the surface
of PLGA to date; however, very few of these appear
promising for soft tissue applications. Plasma treatment,
although very popular,8-11 appears unable to penetrate more
than a few millimeters into the pores of a scaffold. Surface
* To whom correspondence should be addressed. Phone: +61 3 8344
4704. E-mail: jjcw@unimelb.edu.au.

entrapment,6,12-14 while apparently promising for 2-dimensional applications and possibly the denser bone and cartilage
scaffolds, leads to collapse of the thin (<1 m) walls of a
soft tissue scaffold during the swelling step. Partial surface
hydrolysis by acid or base treatment6,15-17 has been much
too aggressive in the past, with conditions that destroy a
typical soft tissue TIPS scaffold2,3 in less than one minute.
A desirable property of any surface modification technique
is that it is inherently limited to the very surface of the
substrate irrespective of reaction rate. As an example, soft
tissue scaffolds for breast reconstruction are somewhat
irregularly shaped, have volumes ranging from 100 mL to 2
L,1 and, in the case of PLGA scaffolds produced via the TIPS
process, have internal wall thicknesses as low as 500 nm.3
In this situation, a relatively slow reaction time on the order
of minutes to hours is desirable to ensure controllable
treatment throughout the scaffold.
In this paper, we report on the use of controlled hydrolysis
to produce carboxylic acid functional groups and aminolysis
to produce primary and secondary amine groups on the
surface of thin PLGA films in a highly controlled manner,
with minimal erosion. In addition, covalent binding of a
model amine-functional macromolecule, chitosan, to the
newly formed functional groups was characterized.
Experimental Section
Materials. Poly(D,L-lactic-co-glycolic acid) (PLGA) with
a lactic acid:glycolic acid ratio of 75:25 and an inherent
viscosity in chloroform of 0.69 dL/g (molecular weight
approximately 100 kDa) was purchased from Birmingham

10.1021/bm0343040 CCC: $27.50

2004 American Chemical Society
Published on Web 01/09/2004

464

Biomacromolecules, Vol. 5, No. 2, 2004

Polymers Inc. Ammonia (LR, 25% aqueous solution),

hydrogen peroxide (LR, 30% aqueous solution), chlorotrimethylsilane (CTMS) (>99%), toluene (HPLC grade),
acetone (HPLC), ethanol (AR, >99.7%), n-hexane (HPLC),
dichloromethane (HPLC), and 2-propanol (iso-propyl alcohol, IPA) were obtained from Merck. N-Aminoethyl-1,3propanediamine (AEPDA) (AR), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), and N-hydroxysuccinimide
(NHS) were obtained from Sigma-Aldrich. Acetic acid (AR),
ethylenediamine (ED) (AR), and triethanolamine (AR) were
obtained from BDH. 2-(N-Morpholino)ethanesulfonic acid
(MES) (AR) was obtained from ICN Biomedicals Inc.
(Ohio). Low molecular weight chitosan (viscosity @ 0.5%
w/v ) 5-20 cP, >80% deacetylated, Wako Fine Chemicals
Inc.) was kindly donated by Professor Yoshinari Baba of
Miyazaki University. All reagents were used as supplied,
and all water used was purified using a MilliPore Simplicity
unit to a resistivity of g18.2 M.cm. All glassware was
washed sequentially in toluene, acetone, and ethanol and
dried using compressed air filtered through activated carbon
immediately prior to use.
Production of Thin Films. PLGA films with a thickness
of <2 m and an RMS roughness of <2 nm measured via
contact-mode AFM imaging over a 10 m 10 m area
were prepared on clean, hydrophobically treated glass
coverslips by a dip-coating method similar to that used by
others.18 Coverslips (22 mm 22 mm, 100 m thick) were
cleaned in a boiling mixture of 9 parts neat hydrogen
peroxide solution and 1 part neat ammonia solution. These
were rinsed in three washes of water, placed individually
into 23 mm internal diameter glass vials so that they stood
vertically, and dried on a hotplate at 80 C. The coverslips
were then made hydrophobic by treatment in a freshly
prepared solution of CTMS in n-hexane (10% v/v) for 10
min and rinsed with three washes of fresh n-hexane, and
allowed to dry by evaporation. These were dipped in a
solution of PLGA in dichloromethane (5% w/v) at 0 C and
withdrawn slowly to leave a thin film of polymer solution
on the glass surface. Most of the solvent evaporated
immediately, leaving a translucent white polymer film on
the glass. The coverslips were then returned to their vials,
loosely capped with aluminum foil and placed horizontally
in an oven at 70 C for 24 h to remove the residual solvent
and anneal the polymer film. At this point the films were
entirely transparent and almost indistinguishable from the
coverslips, except for the presence of refraction fringes,
indicating a very low surface roughness on the order of
nanometers.19
Hydrolysis and Aminolysis of Films. Films to be
hydrolyzed were immersed in a solution of aqueous sodium
hydroxide (NaOH) at suitable concentrations and allowed
to react at 20 C for the desired time period, as described
elsewhere.2 Films to be aminolyzed were immersed in ED
or AEPDA at various concentrations in either water or IPA
and allowed to react at either 20 or 0 C for the desired time
period. Treated films were washed three times with ice cold
water, followed by soaking for a further hour in fresh water
on ice, dried under vacuum over silica gel for 12 h, and stored
in a desiccator over silica gel until further use.

Croll et al.

Activation of Films with Cross-Linking Reagents.

Hydrolyzed films were treated in 10 mL of a freshly made
solution of 4 mM EDAC and 100 mM NHS in 10 mM MES
buffer at pH 6.1 for 1 h at room temperature. Aminolysed
films were treated in freshly prepared 40 mM DMA in 10
mM triethanolamine buffer at pH 8.0 for 1 h at room
temperature. Activated films were rinsed thoroughly with
water and used immediately.
Binding of Chitosan to Films. Chitosan was dissolved
in 0.1 M acetic acid to a concentration of 10 mg/mL. This
solution was filtered through a 0.25 m syringe filter to
remove insoluble particles. A 1 mL aliquot of this solution
was made up to 90 mL with water, and 10 mL of 100 mM
triethanolamine, 1.4 M NaCl at pH 8.0 or 9.0 (adjusted with
1 M NaOH) was added. At this point, the solution turned
slightly milky, indicating the formation of a stable colloidal
suspension of 100 g/mL chitosan with a physiological salt
concentration.
Preactivated surfaces or nonactivated hydrolyzed or aminolyzed controls were incubated in 10 mL of this chitosan
suspension at pH 8.0 (aminolyzed surfaces) or 9.0 (hydrolyzed surfaces) for 1 h. The coated surfaces were then washed
twice with 0.1 M acetic acid to remove all weakly bound
chitosan, followed by three washes with water.
Scanning Electron Microscope (SEM) Analysis. Samples
to be analyzed via SEM were sputter-coated with gold under
vacuum in an argon atmosphere using a sputter current of
60 mA (Dynavac Mini Coater, Dynavac USA). SEM analysis
was carried out with a Philips XL30 SEM using an LaB6
electron gun with an accelerating voltage of 20 kV.
Contact Angle Measurement. Equilibrium advancing (a)
and receding (r) air/water contact angles were measured
with a contact angle goniometer and drop shape analysis
software (First Ten Angstroms, Inc) using the captured
(sessile) drop method20 on PLGA coated glass coverslips at
a temperature of 22.5 C. Water was pumped onto the surface
of the coverslip through a 23-gauge stainless steel needle at
a rate of 2 L/s until the drop diameter was greater than 4
times the diameter of the needle. At this point, the drop was
allowed to stand for 2 min before measuring a. Water was
then removed from the drop at a rate of 2 L/s until the
interface began to recede. A further 2 min relaxation time
was allowed before r was measured. During the first and
second relaxation periods, contact angles decreased or
increased by 3-5 respectively; the contact angles changed
by <0.5 in the next 3 min. Three drops (six interfaces) were
imaged for each surface; values reported are the mean (1
standard deviation.
X-ray Photoelectron Spectroscopy (XPS) Analysis. XPS
analysis was performed using a AXIS HSi spectrometer
(Kratos Analytical Ltd) equipped with a monochromatic Al
KR source at a power of 300 W. The total pressure in the
main vacuum chamber during analysis was typically 2
10-8 mbar. Elements present were identified from survey
spectra. For further analysis, high-resolution spectra were
recorded from individual peaks at 40 eV pass energy. Atomic
concentrations of each element were calculated by determining the relevant integral peak intensities (using a linear type
background) and applying the sensitivity factors supplied by

Hydrolysis and Aminolysis of PLGA

Scheme 1. Methods for Functionalization of PLGA Investigated in
This Papera

a PLGA was first activated by either base hydrolysis or aminolysis, and

further functionalized by covalently binding chitosan using either EDAC
or DMA.

the instrument manufacturer. A value of 285.0 eV for the

binding energy of the main C 1s component (CHx) was used
to correct for charging of specimens under irradiation.
Results and Discussion
Reaction Scheme. The overall route taken to functionalize
PLGA via hydrolysis or aminolysis is shown schematically
in Scheme 1. Chitosan was used as a label to indicate the
activity of the newly formed groups toward covalent binding,
due to its relatively nonadhesive nature compared to proteins.
Hydrolysis. Hydrolysis of polyesters such as PLGA can
be driven by either acidic or basic conditions. Various
mechanisms have been proposed for acid-catalyzed hydrolysis of esters,21 but it is generally agreed that the reaction is
driven via electrophilic attack by hydrogen ions on the
carbonyl oxygen. A number of factors make this scheme
unfavorable for surface-specific modification. The reaction
is driven by small, highly mobile protons, which are able to
diffuse relatively easily between the uncharged polymer
chains and are regenerated by the reaction. Thus, a very fast
reaction rate is required to restrict the hydrolysis to the
surface. Since the ester bonds are only weakly available for
electrophilic hydrolysis, this requires very strongly acidic
conditions. It has been found in the case of poly(L-lactic acid)
(PLLA) that hydrolysis in aqueous solution at pH 2 leads to
bulk, rather than surface hydrolysis.22 Acid hydrolysis of
PLGA for the purposes of surface modification has been
reported before in the literature using extremely strong
perchloric acid/chlorate solutions,5,16,17,23 which led to significant levels of macroscopic degradation, on the order of
tens of microns.
Under basic conditions, hydrolysis is driven via nucleophilic attack by hydroxide ions on the region of lowest
electron density, the carbonyl carbon. These hydroxide ions
are consumed during the hydrolysis reaction, in the process
generating negatively charged carboxylic acid groups on the
polymer surface. The electrostatic repulsion between these
negatively charged end groups and solvated hydroxide ions,

Biomacromolecules, Vol. 5, No. 2, 2004 465

combined with the fact that the hydroxide ion is somewhat

bulkier than the proton and is not regenerated, acts to reduce
the rate of diffusion of hydroxide ions into the polymer. Thus,
under these conditions surface-oriented, rather than bulk,
hydrolysis is favored. Once again, this is borne out by
experimental evidence in the literature both on PLLA22 and
on poly(ethylene terephthalate) (PET) used for textile
manufacture.24,25 On 50 m thick PLLA films hydrolyzed
in aqueous sodium hydroxide solution at pH 12,22 weight
loss showed a linear profile indicating surface erosion,
reaching 70% loss after 150 days, which equates to an
erosion rate of approximately 5 nm per hour on each surface
of the double-sided films, compared to a sigmoidal weightloss profile at pH 2.0, typical of bulk degradation. Base
hydrolysis of PLGA for the purposes of surface modification
has been reported in the past;15,26 however, the authors used
very high concentrations of sodium hydroxide that led to
high levels of surface erosion, with a concomitant increase
in roughness.
Base hydrolysis of model esters such as ethyl acetate and
methyl formate has been found to be first-order in hydroxide
ion concentration,27-29 with a very high activation energy
(105 kJ/mol in the case of ethyl acetate27,28). Thus, the rate
of hydrolysis is strongly dependent on both solution pH and
temperature: a pH change of one unit causes a 10-fold
change in reaction rate, whereas in the case of ethyl acetate,
there is a further 10-fold difference in the rate between 0
and 20 C.
Aminolysis. Aminolysis is a technique which has been
used in the textile industry for many years to improve the
dyeability, wettability characteristics, and feel of synthetic
polyesters such as PET.24,30-33 However, its applicability to
surface modification of biodegradable polyesters for tissue
engineering has only recently been noticed by our group and
others.34-36
A large amount of work has been carried out in the past
to elucidate the mechanism of aminolysis of model
esters.29,37-44 The reaction proceeds via nucleophilic attack
on the carbonyl carbon to form a positively charged
tetrahedral intermediate. Under acidic or neutral conditions,
the amine leaving group (R-NH2) is strongly preferred over
the alcohol leaving group (RC-O-), and hence, the reaction
does not proceed. However, under basic conditions, the
tetrahedral intermediate is deprotonated, leading to the
extremely unfavorable R-NH- leaving group (pKa > 30),
and hence, the reaction proceeds to the formation of an amide
and an alcohol. Therefore, aminolysis is generally carried
out either in basic aqueous solution (pH > pKa of the
amine)29,37,40,42 or in an aprotic, polar solvent with a high
degree of basicity, such as an alcohol.41, 43, 44
Since both the polymer and the amine modifying species
are uncharged under optimal aminolysis conditions, the
reaction is not inherently self-limiting as in the case of
alkaline hydrolysis. It has been shown, for example, that
small, mobile amines such as methylamine and N-propylamine are able to diffuse readily into PET fibers, causing
significant cracking.24,30,33 In addition, weight loss profiles24,30
showed a lag phase after the beginning of treatment typical
of a bulk degradation process.

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Biomacromolecules, Vol. 5, No. 2, 2004

Croll et al.
Table 1. Fitted First-Order Decay Parameters for Treated
Surfaces (Treatment Temperature ) 20 C)
control
0.01 N NaOH
0.05 N NaOH
0.05 M ED (aq)
0.05 M ED (IPA)
0.05 M AEPDA (aq)
0.05 M AEPDA (IPA)

a, eq ()

r, eq ()

t1/2 (min)

74.9 ( 0.6
71.7 ( 0.8
71.6 ( 0.8
70.8 ( 0.7
72.9 ( 0.8
70.1 ( 0.6
71.9 ( 0.8

54.7 ( 0.5
49.1 ( 1.1
48.7 ( 0.4
45.9 ( 0.5
50.2 ( 0.9
44.3 ( 0.5
45.8 ( 0.7

N/A
e1
e 0.2
2.7 ( 0.2
6.0 ( 0.5
1.7 ( 0.2
7.6 ( 0.5

Figure 1. SEM image of a fractured PLGA-coated coverslip. The

thickness of the film here is approximately 1-2 m. G ) glass
substrate, P ) polymer film. Bar ) 10 m.

In contrast to base hydrolysis, the overall activation energy

for the aminolysis reaction is very low and in organic
solvents is often negative,44 leading to a low or inverse
dependence of the reaction rate on temperature. In aqueous
solutions, the reaction rate typically reaches a plateau at pH
values just above the pKa of the amine, dropping sharply at
pH values below this.29,42 It has been found from numerous
experiments39 that in general the log of the aminolysis rate
increases with the pKa of the amine to the 0.8th power.
Surface Characterization. It is well-known that surface
roughness on the micron scale has a strong effect on
measured contact angles,19,20,45 as well as cell-surface
interactions.46 This can be a confounding factor when
attempting to quantify the effect of surface treatments on
these parameters, a fact which appears to have been largely
ignored in much of the literature on surface modification.
SEM and AFM images of many PLGA or polylactic acid
(PLA) films produced by solvent casting from dichloromethane described in the literature display very high levels
of surface roughness, mostly due to the formation of
microscopic bubbles of escaping solvent vapor.6,16,26 By
comparison, the thin films used in this work (Figure 1) are
typically 1-2 m thick, with an RMS roughness of <2 nm
measured by AFM imaging over a 100 m2 area. As can be
seen from Figure 1, the surface undulates slightly over larger
areas; however, these undulations are smoothly varying over
relatively large length scales. Evidence from the literature
indicates that it is sharp changes in topology that have the
most effect on contact angles19,20,47 and cell responses.46,48
These very thin, smooth films allow much more reliable
quantification of surface characteristics such as contact angle
and cell adhesion properties than is achievable using solventcast films.
Contact Angle. As a surface-specific lysis reaction such
as hydrolysis or aminolysis proceeds, the number of active
end groups present at the surface initially increases. However,
eventually an equilibrium is reached at which the rate of
removal of soluble oligomeric fragments is equal to the rate
of generation of new end groups. The density of functional
groups at this point is likely due to a number of factors such
as the initial molecular weight of the polymer, the molecular

Figure 2. Relationship between treatment time and air/water contact

angle for PLGA surfaces treated in (a) 0.01 N NaOH and (b) 0.05 N
NaOH at 20 C. Diamonds ) a, squares ) r. Lines ) fitted firstorder decay profiles.

weight at which oligomers become soluble, and the surface

organization of the polymer.
As may be expected from the mechanism of modification,
the a and r of hydrolyzed or aminolyzed films appeared
to decrease to equilibrium values via a first-order decay
profile. Consequently, first-order decay models were fitted
simultaneously to a and r profiles for each treatment
method. The fitted equilibrium advancing (a,eq) and receding
(r,eq) contact angles and the decay half-time (t1/2) are reported
in Table 1.
For surfaces treated in 0.01 or 0.05 N NaOH (Figure 2),
equilibrium levels of modification were reached extremely
quickly, with decay half-times on the order of seconds. As
expected, the NaOH concentration had very little effect on
the final level of modification; the equilibrium values of a
and r were not significantly different for surfaces treated
in 0.01 N NaOH or 0.05 N NaOH (P > 0.5). Films treated
for up to 24 h in 0.01 N NaOH had slightly reduced a and
r values of 69.7 ( 0.7 and 47.4 ( 0.7, respectively. Films

Biomacromolecules, Vol. 5, No. 2, 2004 467

Hydrolysis and Aminolysis of PLGA

due to the lower catalytic ability of IPA compared to water37

as well as the absence of the concurrent hydrolysis reaction.39,40
To probe the relative contributions of hydrolysis and
aminolysis in aqueous solution and to maximize the amine
yield, PLGA surfaces were treated for 2 h with a series of
solutions of 0.10 or 1.0 M ED at various pH values. To test
the effect of temperature on the reaction selectivity, surfaces
were treated in solutions of identical composition at either
0 or 20 C.
The rate equation for aminolysis of PLGA by ED in
aqueous solution was assumed to be analogous to that
proposed by Satterthwait and Jencks42 for the reaction of
hydrazine with model esters:
ra
[ED]free[ester]

k1 + k2[ED]free + k3[OH - ]

1+

k - 1aH+ + k-2[EDH + ] + k-3

(1)

k-

where ra is the overall aminolysis rate (M min-1), [ED]free

and [EDH+] are the concentrations of free and ionized ED,
respectively, and kn are rate constants.
[ED]free and [EDH+] can be calculated from the Kb of ED
and the pH via
[ED]free ) [ED]tot - [EDH + ] )
Figure 3. Relationship between treatment time and air/water contact
angle for PLGA surfaces treated in (a) 0.05 M ED and (b) 0.05 M
AEPDA at 20 C. Diamonds ) a, squares ) r. Closed symbols )
aqueous, open symbols ) IPA solvent. Lines ) fitted first-order decay
profiles.

treated in 0.05 N NaOH were etched through to the glass

substrate in places after approximately 3 h.
Surfaces treated in 0.05 M ED or AEPDA (Figure 3)
displayed somewhat more interesting behavior. The limiting
values of a and r for surfaces treated with AEPDA in IPA
were significantly lower than those surfaces treated with ED
in IPA (P < 0.05 advancing, P < 0.01 receding). Contact
angles on surfaces treated with aqueous AEPDA were also
slightly lower than on surfaces treated with aqueous ED (P
) 0.1 advancing, P < 0.05 receding). Furthermore, the
limiting contact angles for either treatment in water were
significantly lower than the corresponding values for hydrolysis (P < 0.1 for ED advancing, P , 0.01 for ED
receding and AEPDA). These decreases in contact angle are
likely to be due to extra added hydrophilic groups per
reaction site, rather than a higher overall modification density.
While hydrolysis yields two hydrophilic species per reaction
(a carboxylic acid and an alcohol), ED yields three (amide
bond, primary amine, and alcohol) and AEPDA yields four
(amide, primary amine, secondary amine, and alcohol).
Under aqueous conditions, the limiting contact angles for
both aminolysis treatments were significantly lower than
those reached by aminolysis in IPA (P < 0.01). In IPA
solution, aminolysis is the only reaction able to occur at any
appreciable rate.37 The fitted t1/2 values under these conditions
were somewhat higher than those in water. This is likely

[ED]tot 1 -

Kb
-

[OH ] + Kb

(2)

where [ED]tot is the total concentration of ED in solution.

The competing base hydrolysis reaction is first-order in
hydroxide ion,42 thus
rh
-

[OH ][ester]

) k0

(3)

where rh is the hydrolysis rate (M min-1). The total rate, rt,

is therefore
rt
[ester]

) ra[ED]free + rh[OH - ]

(4)

and the ratio of amine to carboxylic acid groups on the treated

surface can be defined by
)

+1

(5)

where
)

ra[ED]free
rh[OH - ]

(6)

The ratio of the amine group density on the surface, Fa, to

the maximum possible density, Fa,max, is then
Fa
Fa,max

) (1 - e-kt)

(7)

where t is the treatment time and k ) crt/[ester] is the surface

pseudo-first-order decay constant, in units of min-1. The

468

Biomacromolecules, Vol. 5, No. 2, 2004

Croll et al.

Table 2. Fitted Parameters for Kinetic Model of ED Aminolysis in

Aqueous Solution
parameter

units

value
T ) 0 C

value
T ) 20 C

k0
k1a
k2a
k3a
k-1/k-a
k-2/k-a
k-3/k-a
Kb c
q0ac
q0r c
c
qeq
a,h
c
qeq
r,h
a
qeq
a,a
a
qeq
r,a
c
tc

M-1 min-1
M-1 min-1
M-2 min-1
M-2 min-1
M-1
M-1
M-1
dimensionless
degrees
degrees
degrees
degrees
degrees
degrees
dimensionless
min

0.33a
0.032
0.019
0
0
3.62
0
2 10-4
75.0
54.8
71.6
48.8
69.1
44.9
17.3
120b

4.0b
0.15
0.94
0
0
7.06
0
6.4 10-5
75.0
54.8
71.6
48.8
67.6
44.9
17.3
120*

Fitted. b Held fixed. c Measured.

constant c is used to convert from bulk to surface-specific

rates. Although the bulk rate constants are unknown, it was
assumed as a starting point that the bulk rate of hydrolysis
at 20 C is approximately equal to that of ethyl formate.42
Assuming that the measured change in contact angle is a
linear combination of the changes due to hydrolysis and
aminolysis
-kt
-kt
a ) (eq
) + (1 - )(eq
) (8)
a,a + a,ae
a,h + a,he
-kt
-kt
r ) (eq
) + (1 - )(eq
) (9)
r,a + r,ae
r,h + r,he

where the second subscript on refers to aminolysis (a) or

hydrolysis (h), the superscript eq refers to the contact angle
after treatment to equilibrium, and ) 0 - eq refers to
the maximum possible change in contact angle due to
hydrolysis or aminolysis.
For each treatment temperature, the above model was fitted
simultaneously to the pH-dependent data for both 0.1 and 1
M ED concentration using the least-squares method, with
the squared errors weighted by the inverse of the standard
deviation at each data point. In addition, the half-time and
equilibrium contact angles determined for 0.05 M ED at 20
C and its native pH (Figure 3a) were used to constrain the
20 C model fit.
According to the fitted parameters (Table 2), there was
little evidence of catalysis of the reaction by OH- (k3) or of
inhibition by H+ (k-1/k-). Half-times for pure alkaline
hydrolysis at pH 12 (0.01 N NaOH) and pH 12.7 (0.05 N
NaOH) and 20 C were calculated using the fitted values of
k0 and c to be 1.0 and 0.2 min, respectively, in good
agreement with the time-dependent data (Figure 2 and Table
1).
Plots of contact angles versus treatment pH (Figure 4)
displayed three distinct regions. At high pH >12, hydrolysis
is the dominating reaction, and hence, the measured contact
angles approach those expected from pure hydrolysis. At
intermediate pH, a minimum is reached where aminolysis
is the dominant reaction, whereas at low pH, the reaction

kinetics are too slow to give a significant active group density

within the treatment time, and hence, the contact angle
remains close to control levels.
Although the hydrolysis rate dropped markedly with
decreasing temperature, the aminolysis rate was decreased
by a similar amount, giving no useful gain in selectivity
(Figure 5).
The maximum achievable amine group density according
to the model (Figure 6) was greater than 90% of the
maximum possible density for all temperature and concentration conditions tested. At 0 C, however, both the height
and width of the maxima were smaller compared to
equivalent amine concentrations at 20 C, indicating that
lowering of the temperature is actually somewhat detrimental.
The calculated maximum surface amine density as a function
of amine concentration (Figure 7) showed a steep increase
at low concentration, leveling out at approximately 0.05 M
ED for 20 C. At 0.1 M ED, the optimal amine density is
approximately 97% of the maximum; there appears to be
very little gain in increasing the ED concentration further
than this.
XPS Analysis. In the case of partial surface hydrolysis
by NaOH treatment, no detectable changes in surface
composition using XPS are expected. Simple calculations
show that for every 1% of ester bonds hydrolyzed the amount
of oxygen present in the surface should increase by only
0.1 atomic % (at. %). Since the random error in XPS is
approximately 1-2% of the measured value for elements
present in abundance,49 this level of modification is undetectable. Moreover, since the number and type of carbonoxygen bonds are unchanged by hydrolysis (Figure 8), no
change in the relative peak areas in the carbon 1s spectrum
are expected. Although it is true that alcohols and carboxylic
acids display small peak shifts of approximately 0.1 and 0.2
eV relative to ethers and esters respectively,50 in practice
these are inseparable from the existing peaks.
In the case of aminolysis, a new element, nitrogen, is added
to the surface, making detection somewhat easier. Once
again, however, the expected level of nitrogen present is very
small.
The atomic compositions calculated from the XPS widescan spectra for untreated, hydrolyzed, and aminolyzed
surfaces are detailed in Table 3. The measured composition
of untreated films was very close to the theoretical composition of 75:25 PLGA. As expected, hydrolysis or aminolysis
induced very little change in the measured compositions,
except for a slight increase in the carbon:oxygen ratio,
attributed to the CHx peak by analysis of the C1s spectrum
(Figure 9). This change is not significant, however, given
the typical random error in XPS results. The shape of the
C1s spectrum for untreated films was very close to the
expected shape for 75:25 PLGA calculated from the atomic
composition.
PLGA surfaces aminolyzed in aqueous ED or in AEPDA
in IPA for 120 min were found to contain approximately
0.5 at. % nitrogen within the XPS sampling depth of about
5 nm. Thus, calculations based on the empirical formulas of
PLGA, ED, and AEPDA indicate that approximately 1.2 ED
molecules or 0.8 AEPDA molecules were added per 100

Hydrolysis and Aminolysis of PLGA

Biomacromolecules, Vol. 5, No. 2, 2004 469

Figure 4. Contact angle data for surfaces treated with ED in water for 2 h at (a and b) 20 C and (c and d) 0 C, fitted to model described in
eqs 1-9. and - - -, 0.05 M ED; b and s, 0.1 M ED; b and - - - -, 1 M ED.

Figure 5. Calculated rates of aminolysis and alkaline hydrolysis as

a function of pH and temperature. Triangles ) aminolysis ([ED] )
0.1 M), squares ) hydrolysis. Closed symbols ) 20 C, open symbols
) 0 C.

monomer units within this depth. Because the C1s binding

energies for amines (285.5) and amides (288.5) are very
close to the binding energies for CHx and C(O)O, respectively, this level of modification cannot be deconvoluted from
the C1s spectrum.
The level of silicon detected on the tested surfaces was at
or below the detection limit of the XPS instrument, indicating
that the PLGA films formed a cohesive layer completely
covering the glass substrate both before and after treatment.
To study the availability of the newly created surface
groups for covalent binding, low molecular weight chitosan
was used as a naturally nonadhesive amine-functional
biological macromolecule. Two commonly used cross-linking

Figure 6. Ratio of amine groups to the maximum possible group

density on the surface of PLGA treated with ED for 120 min as a
function of pH. Squares ) 1 M ED, triangles ) 0.1 M ED, diamonds
) 0.05 M ED. Closed symbols ) 20 C, open symbols ) 0 C.

systems, EDAC/NHS and DMA, were used to preactivate

hydrolyzed and aminolyzed surfaces, respectively. Samples
were rinsed thoroughly with 0.1 M acetic acid solution after
treatment to ensure that only strongly bound chitosan
remained attached.
The N 1s spectra for these treated surfaces are shown in
Figure 10, and the corresponding nitrogen concentrations are
shown in Table 4. Surfaces with adsorbed chitosan (c) were
essentially indiscernible from the associated hydrolyzed or
aminolyzed surfaces (b), indicating that physical adsorption
of chitosan occurred only to a very low degree on all
surfaces. Preactivation of the surfaces led to distinctly higher
nitrogen concentrations (d). In all cases, the amount of

470

Biomacromolecules, Vol. 5, No. 2, 2004

Croll et al.

Figure 7. Ratio of amine groups to the maximum possible group

density on the surface of PLGA treated with ED for 120 min as a
function of amine concentration. Solid line, T ) 20 C; dashed line,
T ) 0 C.

Figure 8. C 1s photoelectrons detected by XPS for hydrolyzed PLGA

films. 1: CHx (285 eV). 2: C-O (ether or hydroxyl, 287.1 eV). 3:
C(O)O (carboxyl or ester, 289. 1 eV). The number and type of carbon
bonds is unchanged after hydrolysis, and hence, no change in the C
1s spectrum is expected.
Table 3. Atomic Compositions of Untreated and Primary Treated
PLGA as Determined via XPS

theoretical 75:25 PLGA

theoretical PLA
theoretical poly(glycolic acid)
untreated
0.01 N NaOH 120 min
0.05 M ED 120 min (aq)
0.05 M AEPDA 120 min (IPA)

O
(at. %)

C
(at. %)

N
(at. %)

Si
(at. %)

42.2
33.33
50.0
41.1
40.4
39.6
40.8

57.8
66.67
50.0
58.8
59.6
59.9
58.8

0
0
0
<0.1
<0.1
0.5
0.5

0
0
0
<0.1
<0.1
<0.1
<0.1

nitrogen present increased by 0.45-0.6 at. %. The atomic

concentrations of carbon and oxygen also increased and
decreased, respectively, consistent with the presence of
approximately 5 mol % chitosan within the XPS sampling
depth.
General Discussion. The thick polymer films currently
used by many groups for testing of surface modification
techniques are far from an ideal model for most tissue
engineering scaffolds. These films, generally with thicknesses
ranging from tens to hundreds of microns, are generally 1
or 2 orders of magnitude thicker than the fine structures
within such scaffolds. Hence, many treatments which seem
promising when tested on such models involve conditions
which quickly destroy these structures. In addition, thick
films prepared via the popular solvent casting technique with
dichloromethane6,16,26 display surface features on a scale

Figure 9. XPS C1s spectra of untreated and primary treated

samples. (a) Untreated (CHx ) 30.1%), (b) 0.01 N NaOH 120 min
(CHx)31.2%), (c) 0.05 M AEPDA in IPA 120 min (CHx ) 30.0%), (d)
0.05 M ED (aq) 120 min (CHx ) 31.8%). Theoretical CHx ) 27.3%.

which has been shown to strongly influence cell-surface

interactions,46 as well as contact angle measurements.19,20,45
The dip-coating/annealing technique described here results
in films with a thickness of 1-2 m and roughness on the
scale of a few nanometers. These very smooth films allow
much easier determination of the physical and chemical
effects of treatments, while their small thickness demands
stringent control of conditions similar to those required to
avoid structural collapse in a 3-dimensional situation.
Contact angle data showed that the level of modification
quickly reached a limiting value, independent of time and
concentration of modifying species, as expected given the
chain-lysis mechanism of hydrolysis and aminolysis. The
smaller drop in contact angle achieved by aminolysis in IPA
compared to aminolysis in water can be explained by a lower
equilibrium amine group density in the former. This may be
due to increased solubility of PLGA fragments in IPA and
possibly swelling of the molecular structure at the surface,
allowing for easier disentanglement and removal of these
fragments.
Using a relatively simple rate equation (eq 1),42 it was
possible to combine all of the collected contact angle data
for aqueous ED aminolysis and hydrolysis into a single selfconsistent model. The model shows that, due to competition
between aminolysis and hydrolysis at high pH and the
nonlinear relationship between aminolysis rate and pH, the
maximum amine group density for any given ED concentration is achieved at around pH 9.8, where approximately 50%
of the amine is ionized (measured pKa ) 9.8 ( 0.05) and
hence unreactive. Furthermore, the concentration of ED
required to achieve 97% of the theoretical maximum amine
group density is only 0.1 M.
Reducing the temperature of the aqueous aminolysis
reaction from 20 to 0 C results in an approximately 10fold drop in aminolysis rate, and a 12-fold drop in the
hydrolysis rate for any given pH. Although this indicates
that reduced temperature is not a useful option for surface
modification, it indicates that storage in neutral solution at
reduced temperature may be a valid option for PLGA
scaffolds; the calculated half-time for base hydrolysis at pH
7 and 0 C is in excess of two years.
Without the use of further labeling techniques, the XPS
C1s spectrum is of little or no use in analyzing surface

Biomacromolecules, Vol. 5, No. 2, 2004 471

Hydrolysis and Aminolysis of PLGA

Figure 10. XPS N 1s spectra of surfaces modified by (i) 0.01 N NaOH for 120 min, (ii) 0.05 N NaOH for 30 min, (iii) AEPDA in IPA for 120 min,
(iv) aqueous ED for 120 min. (a) Untreated, (b) primary treated, (c) primary + adsorbed chitosan, (d) primary + covalently bound chitosan.
Table 4. Nitrogen Content of Films after Treatment and Chitosan
Attachment
treatment

primary
(at. %)

primary + ads.
(at. %)

primary + cov.
(at. %)

untreated
0.01 N NaOH
0.05 N NaOH
0.05 M AEPDA
0.05 M ED

<0.1
<0.1
<0.1
0.5
0.5

N/A
0.15
0.1
0.4
0.5

N/A
0.45
0.65
1.1
1.05

modifications of this type on ester-based polymers, because

the very small changes caused are obscured by the underlying
polymer peaks. The slight enrichment of CHx found in the
surface of most treated samples is not significant enough to
draw any conclusions.
The amount of chitosan attached to the surfaces via
covalent binding was somewhat lower than that reported
using surface entrapment modification of PLLA.14 However,
surface entrapment does not appear to be applicable to fine
three-dimensional structures.
Although many groups working on surface modification
in the past have aimed for very high levels of surface reactive
groups on substrates such as PLGA, a simple analysis of
the scale factors involved shows that this may not be
necessary. Based on its crystal structure,51,52 human serum
albumin, a relatively small protein, covers an area of
approximately 100 nm2, equivalent to roughly 400-600
PLGA monomer units, and hence, only a very low level of
primary modification (<1%) is theoretically required to allow
the attachment of a complete monolayer of protein via

covalent binding. The data presented here shows that the

level of surface coverage with active species achieved by
hydrolysis or aminolysis is at least 1%, and possibly as high
as 5%, which appears more than adequate for the purpose
of further functionalization.
Concluding Remarks
Creation of active groups allowing covalent attachment
of further species on the surface of a polymer such as PLGA
is a necessary first step toward the creation of a truly
biomimetic polymer-based scaffold for tissue engineering.
Various methods for creation of these groups have been
proposed in the past; however, these have generally displayed
major problems in translating to a 3-dimensional structure
of arbitrary size, shape, and structure. Soft tissue scaffolds
in particular generally have large volumes, complex shapes
and internal wall structures of submicron dimensions.
In this study, we have demonstrated the use of two
different mild wet chemical techniques, controlled surface
hydrolysis and aminolysis, to activate the surface of poly(lactic-co-glycolic acid) (PLGA) with either carboxylic acid
or primary amine groups. Using contact angle measurements,
we have shown that the concentration of these active groups
on the surface quickly reaches an equilibrium value independent of the modifying species concentration. It was shown
that, in aqueous solutions of ethylenediamine (ED), conditions can be chosen under which the contribution of
hydrolysis to the surface modification is negligible, even at
remarkably low concentrations of ED.

472

Biomacromolecules, Vol. 5, No. 2, 2004

The amine-functional polysaccharide chitosan is regarded

by many as a candidate biomaterial in its own right;53-55
however, we have used it here simply as a normally
nonadhesive model biomacromolecule for covalent binding
studies. X-ray photoelectron spectroscopy (XPS) analysis
showed that carboxyl- or amine-functionalized surfaces were
readily available for covalent binding of chitosan using
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC)/
N-hydroxysuccinimide (NHS) or dimethyl adipimidate (DMA),
respectively.
The PLGA films used as model surfaces throughout this
study were no more than 2 m thick, with RMS roughness
values of <2 nm on untreated films. Although films treated
in 0.01 N NaOH reached equilibrium contact angle values
within 5 min, they were still intact after 24 h, indicating an
extremely slow etching rate. Hence, these treatments may
be carried out within even the most delicate porous structures,
with little loss of mechanical strength.
To direct tissue growth into a tissue engineering scaffold,
it is not sufficient to simply provide a surface on which cells
grow well. Equally important is the retention of the phenotype of the tissue. To control this, the surface of the scaffold
should ideally resemble as closely as possible the natural
extracellular matrix of the desired tissue. Thus, specific and
permanent binding of proteins to the scaffold surface appears
to be a necessity. The modifications described here have been
shown to allow this, using chitosan as a model, nonadhesive
biomolecule. Furthermore, since the reaction rates are tunable
over a wide range, there is no theoretical limit to the size or
shape of scaffolds that can be treated via this method.
Acknowledgment. The authors thank CSIRO Molecular
Science for kindly allowing the use of their laboratory and
XPS facilities during this work. Thanks also to Dr. David
Steele for his useful discussions and advice and to Roger
Curtain for his expert help with the SEM. We also gratefully
acknowledge the funding support of the Australian Research
Council and the Particulate Fluids Processing Centre.
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