Degradation mechanism and kinetics of thermosensitive polyacrylamides containing lactic acid side chains

Article abstract of DOI:10.1021/ma034381n

Diblock copolymers of poly(N-isopropylacrylamide-co-N-(2-hydroxypropyl)methacrylamide lactate) (poly(NIPAAm-co-HPMAm-lactate)) as a thermosensitive block and poly(ethylene glycol) (PEG) as a hydrophilic block form polymeric micelles above the cloud point (CP) of the temperature-sensitive block. Destabilization of these micelles occurs upon hydrolysis of the lactate side chains. Here we report on the degradation kinetics of the HPMAm-mono(di)lactate monomers and their copolymers with NIPAAm. The degradation of the monomers and polymers in their soluble state (thus below their CP) followed normal ester hydrolysis behavior: the degradation rate increased with temperature, pH (from pH 7.5 to 11), and dielectric constant of the medium. Above the CP, where the polymers are in a precipitated state, a significant retardation of the polymer degradation occurred due to a decrease of dielectric constant of the local environment of the precipitated polymer. This study shows that it is possible to predict the rate of formation of HPMAm in NIPAAm-co-HPMAm-lactate copolymers with results in an increase of the overall hydrophilicity of the polymers and destabilization of polymeric micelles based on poly(NIPAAm-co-HPMAm-lactate).

Full text of DOI:10.1021/ma034381n

Macromolecules 2003, 36, 7491-7498
7491
Degradation Mechanism and Kinetics of Thermosensitive
Polyacrylamides Containing Lactic Acid Side Chains
D. Ner a d ovic, M. J . va n Steen ber gen , L. Va n steela n t, Y. J . Meijer ,
C. F . va n Nostr u m , a n d W. E. Hen n in k *
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of
Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands
Received March 26, 2003; Revised Manuscript Received J uly 10, 2003
ABSTRACT: Diblock copolymers of poly(N-isopropylacrylamide-co-N-(2-hydroxypropyl)methacrylamide
lactate) (poly(NIPAAm-co-HPMAm-lactate)) as a thermosensitive block and poly(ethylene glycol) (PEG)
as a hydrophilic block form polymeric micelles above the cloud point (CP) of the temperature-sensitive
block. Destabilization of these micelles occurs upon hydrolysis of the lactate side chains. Here we report
on the degradation kinetics of the HPMAm-mono(di)lactate monomers and their copolymers with NIPAAm.
The degradation of the monomers and polymers in their soluble state (thus below their CP) followed
normal ester hydrolysis behavior: the degradation rate increased with temperature, pH (from pH 7.5 to
11), and dielectric constant of the medium. Above the CP, where the polymers are in a precipitated state,
a significant retardation of the polymer degradation occurred due to a decrease of dielectric constant of
the local environment of the precipitated polymer. This study shows that it is possible to predict the rate
of formation of HPMAm in NIPAAm-co-HPMAm-lactate copolymers which results in an increase of the
overall hydrophilicity of the polymers and destabilization of polymeric micelles based on poly(NIPAAm-
co-HPMAm-lactate).
In tr od u ction
and graft copolymers composed of a hydrophilic chain
(e.g., poly(ethylene glycol) (PEG) or dextran) and a
thermosensitive PNIPAAm chain form temperature-
sensitive polymeric micelles, meaning that below the CP
of PNIPAAm these polymers are soluble in water
whereas they organize themselves in self-assembled
structures (micelles) due to collapse of the PNIPAAm
block above this temperature.26-30 These colloidal par-
ticles, however, do not destabilize at physiological
temperature. Therefore, we recently described a novel
system based on diblock copolymers of poly(NIPAAm-
co-HPMAm-lactate) and PEG.20 These polymers self-
assemble into nanosized particles at physiological tem-
perature. During hydrolysis of the lactate acid side
groups the cloud point increases, resulting in destabi-
lization of the particles once the cloud point of the
thermosensitive block passes 37 °C. It is expected when
the particles are loaded with a drug, this destabilization
process will be associated with release of the drug.
Therefore, it is very important to have insight into the
degradation kinetics of the polymers.
During the past two decades, thermosensitive poly-
mers have been intensively investigated as candidate
biomaterials and drug delivery systems.1-6 Among
them, poly(N-isopropylacrylamide) (PNIPAAm) and its
(block) copolymers have been the most extensively
studied materials.7-13 PNIPAAm is known for its unique
thermally reversible solubility in aqueous solutions. It
exhibits a lower critical solution temperature (LCST)
behavior in water; i.e., it is soluble in water at temper-
atures lower than its cloud point (CP), but this polymer
precipitates above this temperature.14-17 This reversible
hydration-dehydration behavior occurs in a narrow
temperature range (30-33 °C in water).18
In a previous paper,19 we reported the synthesis of
copolymers containing NIPAAm and the lactic esters of
(2-hydroxyethyl)methacrylic acid (HEMA-lactate), a new
type of thermosensitive polymer with hydrolyzable
lactate ester side groups. These random copolymers
have a CP below that of PNIPAAm, which increases due
to the hydrolysis of the hydrophobic lactate ester side
groups. The resulting polymers poly(NIPAAm-co-
HEMA) have a final CP independent of their composi-
tion of approximately 31 °C. Polymers whose CP would
pass 37 °C (body temperature) during hydrolysis of the
side groups would be much more attractive for the
development of for example new drug delivery systems.
We therefore synthesized a thermosensitive copolymer
of NIPAAm and N-(2-hydroxypropyl)methacrylamide
lactate (HPMAm-lactate). Poly(NIPAAm-co-HPMAm-
lactate) with g35 mol % HPMAm-lactate has a CP well
below body temperature and converts into poly(NIPAAm-
co-HPMAm) with a CP above 37 °C upon hydrolysis.20
Amphiphilic block copolymers form polymeric micelles
in aqueous solution.21-25 It has been shown that block
In this paper we report on a detailed study of the
degradation kinetics of HPMAm-di/monolactate mono-
mers and their copolymers with NIPAAm.
Exp er im en ta l Section
1. Ma ter ia ls. 1,4-Dioxane (99+%, Fluka Chemie AG) was
purified by distillation. The other chemicals were used as
received. L-Lactide ((3S-cis)-3,6-dimethyl-1,4-dioxane-2,5-di-
one, >99.5%) was obtained fromPurac Biochem BV (Gorinchem,
The Netherlands). R,R′-Azoisobutyronitrile (AIBN), poly-
(ethylene glycol) 5000 monomethyl ether (PEG 5000), and
dimethyl sulfoxide (DMSO) (puriss) were from Fluka Chemie
AG (Buchs, Switzerland), and N-(2-hydroxypropyl)methacry-
lamide (HPMAm) was from Polysciences, Inc. (Brunschwig
chemie, Warrington, PA). Stannous 2-ethylhexanoate (ap-
proximately 95%, SnOct2) was from Sigma Chemical Co. (St.
Louis, MO), and 4-methoxyphenol (99%) was from J anssen
Chimica (Geel, Belgium). N-Isopropylacrylamide (NIPAAm,
* To whom correspondence should be addressed: tel +31-30
2536964, fax +31-302517839; e-mail W.E.Hennink@pharm.uu.nl.
10.1021/ma034381n CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/06/2003

7492 Neradovic et al.
Macromolecules, Vol. 36, No. 20, 2003
F igu r e 1. Structure of N-(2-hydroxypropyl)methacrylamide
lactate (HPMAm-lactate).
F igu r e 2. Structure of NIPAAm-co-HPMAm-lactate copoly-
mers.
97%) was obtained from Aldrich-Chemie (Steinheim, Ger-
many).
Lactoyl lactate was synthesized, as will be reported in detail
elsewhere.31 In short, lactide was reacted with sec-phenethyl
alcohol to yield sec-phenethyl lactoyl lactate. Next, this
compound was converted quantitatively into the linear dimer
of lactic acid, lactoyl lactate.
MAm-mono(di)lactate (molar ratio 65/35) as thermosensitive
block and PEG as hydrophilic block were prepared by radical
copolymerization via a macroinitiator route.20
1H NMR for the dilactate containing polymer (CDCl3): δ
6.62 (b, H3, H9), 5.35-4.8 (b, H13, H15), 4.38 (b, H12), 3.96 (b,
H4), 3.76-3.39 (b, PEG methylene protons CH2-CH2), 3.7-
2.75 (b, H10), 2.39-0.45 (the rest of the protons). The NIPAAm/
HPMAm-lactate comonomer ratio (mol/mol) in the copolymer
was determined as described above. The number-average
molecular weight (Mn) of the thermosensitive block was
determined by 1H NMR as follows: (a) the value of the integral
of the PEG protons divided by 454 (average number of protons
per one PEG 5000 chain) gave the integral value for one PEG
proton, and (b) the number of NIPAAm units in the polymers
was determined from the ratio of the integral of the methine
proton (H4) of NIPAAm to the integral of one PEG proton. The
ratio of the integral of the methine (H12) proton of HPMAm to
the integral value per one PEG proton gave the number of
HPMAm-lactate units in the polymer. The number-average
molecular weight of the NIPAAm-blocks was calculated from
the resulting number of units.
2. Meth od s. 2.1. 1H NMR Sp ectr oscop y. 1H NMR spectra
were recorded with a Gemini 300 MHz spectrometer (Varian
Associates Inc. NMR Instruments, Palo Alto, CA). Spectra
were obtained with or without TFAA (trifluoroacetic anhy-
dride, 99+%) as a shifting reagent. With DMSO-d6 as the
solvent the central line of DMSO at 2.50 ppm was used as the
reference line. With CDCl3 as the solvent the CHCl3 signal at
7.26 ppm was used as the reference line.
2.2. Syn th esis of N-(2-Hyd r oxyp r op yl)m eth a cr yla m id e
La cta te (HP MAm -La cta te). HPMAm grafted with oligo-
lactate (further abbreviated as HPMAm-lactate) was synthe-
sized according to a slightly modified procedure previously
reported.32,33 In brief, a mixture of HPMAm (4.30 g, 30 mmol),
L-lactide (2.16 g, 15 mmol), and 4-methoxyphenol (a polymer-
ization inhibitor, 0.3 mmol) was heated to 130 °C until the
lactide was molten. Subsequently, a catalytic amount of
stannous octoate (SnOct2, 0.3 mmol) was added. The resulting
mixture was stirred for 5 h at 130 °C and thereafter allowed
to cool to room temperature.
2.5. Sta tic Ligh t Sca tter in g (SLS). Static light scattering
was measured as described previously.19 In short, the copoly-
mers were dissolved in 0.1 M PBS (pH ) 7.2) at a concentra-
tion of 1 mg/mL. Onsets on the x-axis, obtained by extrapola-
tion of the intensity-temperature curves to intensity zero,
were considered as the cloud points (CP). The reported values
are the averages of at least four measurements.
HPMAm-mono(di)lactate was isolated from the crude reac-
tion mixture by preparative HPLC. Therefore, 1 g of the
polydisperse product was dissolved in 200 µL of acetonitrile
and then diluted with 800 µL of water. The resulting solution
was centrifuged at 13 000 rpm for 5 min. After filtration
through 0.45 µm polypropylene filters (Poly Pure filters 4 mm,
Alltech), the solution was applied onto a preparative HPLC
column.34 A gradient was run from 80% A (water/acetonitrile
) 5/95 (w/w)) to 100% B (water/acetonitrile ) 95/5 (w/w)) in
40 min. The flow rate was 10 mL/min. UV detection at a
wavelength of 250 nm was applied, and the individual peaks
were collected. The collected products were freeze-dried and
2.6. Degr a d a tion of th e Mon om er s. Degradation of the
monomers was investigated in 100 mM phosphate buffered
saline (PBS), pH 7.5; the ionic strength (µ) was adjusted to
0.3 with NaCl. For other pH values, bicarbonate buffer (pH
8-9.2) and carbonate buffer (pH 10-11) were used (100 mM).
For each study the pH of the buffer was adjusted at the
temperature at which the study was conducted; the change in
pH was less than 0.1 during the degradation studies. A 0.5
mL stock solution of HPMAm-mono(di)lactate in DMSO (2
mM) was diluted with 4.5 mL of buffer. Degradation reactions
were carried out in glass bottles at various temperatures
ranging from 15 to 60 °C. Samples (300 µL) were periodically
drawn from the reaction mixtures and diluted with 700 µL of
1 M acetate buffer (pH 3.4) in order to stop further degrada-
tion. The samples were stored at 4 °C prior to HPLC analysis
(see below). For each degradation curve at least six samples
were drawn in a time frame corresponding with 1-3 half-lives.
The interday variation in kobs for HPMAm-dilactate was
around 6% (kobs at 60 °C and pH 7.5 was (1.53 ( 0.10) × 10-4
s-1 (n ) 5)).
2.7. Degr a d a tion of th e P olym er s. Degradation of poly-
(NIPAAm-co-HPMAm-monolactate) and poly(NIPAAm-co-HP-
MAm-dilactate) was investigated between 4 and 60 °C. Samples
of 100 µL of the polymer solutions in water (10 mg/mL) were
diluted with 100 µL of 300 mM sodium bicarbonate buffer of
varying pH’s (8, 9, and 10). The pH of the buffer was adjusted
at the temperature of the study. At different time points,
samples were taken, and 10 µL of 4 M HCl was added to adjust
the pH to ∼4 to stop further degradation. The samples were
stored at -20 °C prior to the HPLC analysis (see below).
1
analyzed by H NMR and by RP-HPLC (purity > 98%).
1H NMR (CDCl3) (see Figure 1): HPMAm-monolactate: δ
6.30 (b, 1H, H3), 5.63 (s, 1H, H1), 5.30 (s, 1H, H1′), 5.09 (q, 1H,
H8), 4.24 (q, 1H, H5), 3.55-3.35 (m, 2H, H4), 1.92 (s, 3H, H2),
1.35 (d, 3H, H7) and 1.28 (d, 3H, H6). HPMAm-dilactate: δ
6.25 (b, 1H, H3), 5.70 (s, 1H, H1), 5.35 (s, 1H, H1′), 5.2-5.0 (m,
2H, H8, H9), 4.35 (q, 1H, H5), 3.8-3.3 (m, 2H, H4), 1.92 (s, 3H,
H2), 1.50 (d, 6H, H7, H10) and 1.27 (d, 3H, H6).
3. Syn t h esis of P oly(NIP AAm -co-H P MAm -la ct a t e).
NIPAAm and HPMAm-mono(di)lactate were dissolved at a
total monomer concentration of 0.1 g/mL in 1,4-dioxane. The
monomer feed ratio was NIPAAm/HPMAm-mono(di)lactate )
65/35 (mol/mol). AIBN dissolved in 1,4-dioxane was added
(total amount of monomers/initiator ) 250/1 mol/mol), and the
copolymerization was conducted at 60 °C for 20 h in a nitrogen
atmosphere. Next, the solvent was removed under reduced
pressure, and the copolymers were dissolved in acetone
(around 20% (w/v)) and precipitated in an excess of diethyl
ether. The precipitated polymers were isolated by filtration
and dried in a vacuum oven at 40 °C.
1H NMR for the dilactate containing polymer (CDCl3; Figure
2): δ 6.55 (b, H3, H9), 5.35-4.8 (H13, H15), 4.41 (b, H12), 3.95
(b, H4), 3.42-3.75 (b, H10), 2.62-0.6 (other protons). The
comonomer ratio (mol/mol) in the copolymer was determined
from the ratio of the integral of the methine proton (H4) of
NIPAAm to the integral of the methine (H12) proton of
HPMAm (see Figure 2).
To determine the maximum amount of lactic acid which can
be formed, hydrolysis was forced to completion by adding an
equal volume of 0.1 M NaOH to a polymer solution followed
by incubation at 60 °C for 17 h. The resulting reaction mixture
was neutralized by adding 12.5 µL of 4 M HCl and stored at
-20 °C prior to the HPLC analysis (see below).
2.4. Syn th esis of P oly(NIP AAm -co-HP MAm -m on o(d i)-
la cta te)-b-P EG 5000. Block copolymers with NIPAAm/HP-

Macromolecules, Vol. 36, No. 20, 2003
Thermosensitive Polyacrylamides 7493
Degradation of lactoyl lactate (concentration 1 mg/mL) was
carried out at different temperatures (8-60 °C) and at pH 10.
Analysis of the resulting degradation products was carried out
under the same conditions as described for the polymers.
Degradation of poly(NIPAAm-co-HPMAm-mono(di)lactate)-b-
PEG 5000 was investigated at pH 10 and at 60 °C the same
way as described above.
2.8. Degr a d a tion in Med ia w ith Differ en t Dielectr ic
Con sta n ts. Samples (60 µL) of stock solution of HPMAm-
dilactate in DMSO (10 mM) were diluted to 3 mL with DMSO/
sodium bicarbonate buffer mixtures. DMSO to buffer ratios
of 10:90, 20:80, 30:70, 40:60, and 50:50 (v/v) were used
(dielectric constant (ꢀ) ranges from 76.8 to 63.4). Degradation
was followed at 37 °C and at pH 9.2. The pH of every DMSO/
buffer mixture was adjusted at the temperature of the study.
Samples for the HPLC analysis were prepared the same way
as described above.
2.9. Rever sed -P h a se High -P er for m a n ce Liqu id Ch r o-
m a togr a p h y (RP -HP LC). HPLC analysis of the samples was
carried out on a Waters system (Waters Associates Inc.,
Milford, MA) consisting of a pump model 600, an autoinjector
model 717, and a variable wavelength absorbance detector
model 486. For the degradation studies of the monomers an
analytical column LiChrosphere 100 RP-18 (5 µm, 125 × 4 mm
i.d.) was used. For the degradation studies of the polymers an
Alltech Prevail organic acid column (5 µm, 250 × 4.6 mm) was
used. The flow rates were 1.0 mL/min, and the detection
wavelength was 210 nm.
A gradient system was run for the analysis of HPMAm-
mono(di)lactate, using methanol/water ) 5/95 (w/w), pH 2
(eluent A) and methanol/water ) 95/5 (w/w), pH 2 (eluent B).
The pH of both eluents was adjusted by addition of perchloric
acid. The gradient was run from 100% A to 100% B in 26 min.
The injection volume was 100 µL. Calibration curves were
generated for each monomer and its degradation products with
freshly prepared standard solutions in 10 mM HClO4/H2O (pH
2.4) in the appropriate concentration range. Peak areas were
determined with Millennium 32 version 3.0 software (Waters
Associates Inc.). The calibration curves for HPMAm-mono(di)-
lactate and HPMAm were linear between 0.2 and 20 nmol.
For analysis of the amounts of lactic acid and lactoyl lactate,
25 mM KH2PO3 (pH 2.5) was used as eluent A and eluent B
was acetonitrile/water ) 95/5 (w/w). A gradient was run from
0% B to 60% B in 10 min, followed for 10 min by elution with
60% B. The injection volume was 50 µL. The calibration curves
for lactic acid and lactoyl lactate were linear between 0.01 and
4 µmol.
F igu r e 3. Degradation profiles of (A) HPMAm-monolactate
and (B) HPMAm-dilactate at 60 °C at pH 7.5 (symbols
represent the experimental data; drawn lines are fitted ac-
cording to eqs 3-5): (2) HPMAm-dilactate; (9) HPMAm-
monolactate; (b) HPMAm.
measurements. In detail, the block copolymer was dissolved
in water (2 mg/mL) at 4 °C (below the cloud point). The
aqueous polymer solution was diluted with an equal volume
of 0.3 M NaHCO3 buffer pH 10 at 4 °C. Before the analysis
the sample was filtrated through 0.4 mm nylon filters (Alltech
Associates Inc., Deerfield, IL). A cuvette with the cold polymer
solution was transferred to the heated cell holder of a spec-
trofluorometer at 37 °C. The light scattering by the polymer
solution at 37 °C was measured in time at a wavelength of
650 ( 2.5 nm under a 90° angle. In a separate experiment,
the particle size in time was monitored using DLS at 37 °C
and at both pH 5 and pH 10. For the study at pH 5, 1 mg/mL
of the polymer was dissolved in 0.15 M ammonium acetate
buffer at 4 °C. The DLS cuvette with the copolymer solution
was cooled to 4 °C and subsequently placed in the heated cell
holder at 37 °C.
2.10. Hyd r olysis of th e Ester Bon d betw een th e P EG
a n d NIP AAm Block s in th e Block Cop olym er . The block
copolymer of NIPAAm and PEG 2000 (6800:2000 g/mol) was
prepared by radical copolymerization using (PEG 2000)2-
ABCPA as initiator essentially as described previously.20 The
hydrolytic degradation of the ester bond between the blocks
Resu lts
Degr a d a tion of th e Mon om er s. The hydrolysis of
HPMAm-mono(di)lactate was determined in the range
15-60 °C and at pH 7.5. At all temperatures the
concentration of both monomers decreased according to
pseudo-first-order kinetics. Typical degradation profiles
of HPMAm-monolactate and HPMAm-dilactate at 60 °C
and pH 7.5 are shown in parts A and B of Figure 3,
respectively. The detected hydrolysis product of HP-
MAm-monolactate was HPMAm, while degradation of
HPMAm-dilactate resulted in the formation of HPMAm-
monolactate and HPMAm. In both cases no MAA was
detected as a degradation product, which can be at-
tributed to the high stability of the amide bond.
in the PNIPAAm(M )6800)/PEG 2000 polymer was followed at
n
37 °C and at pH 8.5. For each study (n ) 4), 100 µL polymer
samples in water (concentration 40 mg/mL) were pipetted into
the Eppendorf vials. Next, 900 µL of 0.1 M Tris buffer (pH
8.5) was added. The vials were placed in a water bath at 37
°C. At different time points vials were taken out of the water
bath, and the pH was adjusted to ∼4 by addition of 18.5 µL of
4 M HCl. The samples were stored at 4 °C prior to analysis.
The formation of PEG 2000 in time was followed by gel
permeation chromatography (GPC) using a model 600 HPLC
pump, a model 410 differential refractometer (Waters Associ-
ates Inc., Milford, MA), and a PL-aquagel-OH mixed column
(8 µm, Polymer Laboratories Inc., Amherst, MA). Filtered 0.1
M Tris buffer (pH 7.5) was used as the mobile phase. The
analysis was conducted at 30 °C. The flow rate was 1 mL/min.
For calibration of the column solutions of PEG 2000 monom-
ethyl ether in eluent with known concentrations were used.
2.11. P r ed ictive Va lu e of th e Kin etic Mod el. To dem-
onstrate the predictive value of the kinetic model, the desta-
bilization of nanoparticles based on poly(NIPAAm-co-HPMAm-
dilactate (65/35 (mol/mol))-b-PEG 5000 at 37 °C and pH 5 and
pH 10 was followed by dynamic and static light scattering
The observed first-order reaction rate constants (kobs
)
at 37 °C and pH 7.5 are for HPMAm-monolactate (2.20
( 0.06) × 10-6 s-1 (t1/2 ) 87.5 h) and for HPMAm-
dilactate (1.25 ( 0.07) × 10-5 s-1 (t1/2 ) 15.4 h).
The pH dependence of kobs for both HPMAm-mono-
lactate and HPMAm-dilactate was studied in the pH
range 7.5-11. kobs can be described by the general rate
constant equation (eq 1):
kobs ) k0 + kH[H+] + kOH[OH-]
(1)

7494 Neradovic et al.
Macromolecules, Vol. 36, No. 20, 2003
F igu r e 4. Influence of the dielectric constant (ꢀ) on kobs (pH
9.2, 37 °C).
F igu r e 5. Arrhenius plots for degradation of HPMAm-mono-
(di)lactate at pH 7.5.
Sch em e 1. P ossible Degr a d a tion Rou tes for th e
HP MAm -Mon o(d i)la cta te Mon om er s (R Is HP MAm )
degradation of HPMAm-monolactate. The fraction of
molecules, which follows the route via the monolactate
and not the one shown on the right side of Scheme 1, is
equal to k1/(k1 + k2). Therefore, for the situation
represented by Scheme 1, eq 2 becomes
k1
k1
(e-k t - e-k t
) (3)
1
3
[M-L]t )
[M-LL]0
k1 + k2
k3 - k1
From the decreasing amounts of HPMAm-dilactate in
time an “overall rate constant” was determined (kobs
that equals the sum of two reaction rate constants k1 +
k2. The values of k1 and k2 were calculated by fitting
the amount of monolactate (M-L) as a function of time
using eq 3.
)
The measured amounts of HPMAm-dilactate [M-LL]t
and HPMAm ([M]t) were fitted to eqs 4 and 5:
Ta ble 1. Rea ction Ra te Con sta n ts for HP MAm -Dila cta te
a t 37 °C (p H 7.5) in Wa ter w ith 10% DMSOa
[M-LL]t ) [M-LL]0e-(k +k )t
(4)
(5)
1
2
monomer
k1 (h-1) ( SD
0.032 ( 0.002
k2 (h-1) ( SD
0.014 ( 0.003
k3 (h-1) ( SD
HPMAm-
dilactate
0.0079 ( 0.0002
[M]t ) [M-LL]0 - [M-LL]t - [M-L]t
a
k1, k2, and k3 refer to the reactions of Scheme 1.
The calculated values for the reaction rate constants
(Table 1) correspond well with the experimental data,
as can be seen in Figure 3A,B.
By following the degradation of lactoyl lactate at
different temperatures and at pH 10, the reaction rate
constant k4 as a function of temperature was deter-
mined.
In this equation k0 is the first-order rate constant for
the degradation in water only, while kH and kOH are
second-order rate constants for degradation catalyzed
by protons and hydroxyl ions, respectively. From the log
kobs-pH graphs for both HPMAm-monolactate and
HPMAm-dilactate, it was concluded that in the pH
range studied (pH 7.5-11) the slopes of the curves for
both monomers approach +1 (results not shown), indi-
cating that the hydrolysis of both monomers in the pH
range investigated is specifically hydroxyl-catalyzed.
This is in agreement with the results reported for other
esters.35-37
The degradation rate of HPMAm-dilactate decreased
with decreasing dielectric constant of the medium
(Figure 4), as reported before for other esters.36-38
The two possible degradation routes of HPMAm-
dilactate are shown in Scheme 1. The route shown on
the left side of Scheme 1 is a consecutive first-order
reaction of the formation and degradation of the mono-
lactate. For this part of the reaction sequence the
following rate equation is valid for the amount of
monolactate present in the reaction mixture:39
The curve fittings were performed with the experi-
mental data at every incubation temperature of HP-
MAm-mono(di)lactate (pH 7.5). Figure 5 shows the
corresponding Arrhenius plot for the hydrolysis of
HPMAm-mono(di)lactate. This figure shows that the
activation energies for the three subreactions are equal
(97 ( 5 kJ /mol).
Degr a d a tion of th e P olym er s. HPMA-monolactate
and dilactate were used to prepare random copolymers
with NIPAAm. NIPAAm/HPMAm-mono(di)lactate mono-
mer feed ratios of 65/35 (mol/mol) were used. The
copolymers were obtained in good yields (80-90%). As
determined by 1H NMR, the monomer ratio in poly-
(NIPAAm-co-HPMAm-monolactate) was 68/32 (mol/mol)
and for poly(NIPAAm-co-HPMAm-dilactate) it was 69/
31 (mol/mol). The cloud points were 31.5 and 18.7 °C,
respectively; after hydrolysis the CP of the formed poly-
(NIPAAm-co-HPMAm) was 43.5 °C.
k1
1
3
Two thermosensitive block copolymers, namely poly-
(NIPAAm-co-HPMAm-monolactate)-b-PEG 5000 and
poly(NIPAAm-co-HPMAm-dilactate)-b-PEG 5000, were
synthesized. The Mn of the thermosensitive block in the
first polymer was 14 500, and the NIPAAm/HPMAm-
monolactate ratio was 68/32 (mol/mol). Poly(NIPAAm-
[M-L]t ) [M-LL]
(e-k t - e-k t
)
(2)
0 k3 - k1
where [M-LL]0 is the starting amount of HPMAm-
dilactate and k1 and k3 are the rate constants as shown
in Scheme 1. k3 equals the before-mentioned kobs of the

Macromolecules, Vol. 36, No. 20, 2003
Thermosensitive Polyacrylamides 7495
F igu r e 7. Arrhenius plot for poly(NIPAAm-co-HPMAm-mono-
(di)lactate) at pH 10. CP1 and CP2 refer to the cloud points of
the polymers with HPMAm-monolactate and HPMAm-dilac-
tate before hydrolysis of the lactic acid side groups, respec-
tively.
Equation 7 is derived knowing that the amount of free
lactic acid ([L]t) is equal to 2 times the amount of
polymer-bound dilactate at t ) 0 minus the amount of
lactic acid still bound to the polymer or present as its
dimer at time t. The total amount of lactic acid in the
copolymer at t ) 0 (2[P-LL]0) is known from the
composition of the polymer as determined by NMR (see
above) and was checked by HPLC determination of the
amounts of lactic acid produced after driving the hy-
drolysis to completion using 0.1 M NaOH at 60 °C.
Combining eqs 3, 4 and 6 with eq 7 yield
F igu r e 6. Degradation curves of (A) poly(NIPAAm-co-HP-
MAm-monolactate) at 25 °C and at pH 10 and (B) poly-
(NIPAAm-co-HPMAm-dilactate) at 60 °C and at pH 10 (sym-
bols are experimental data; drawn lines are calculated by curve
t
fitting according to the equation [L]t ) [P-L]0e-k for the
3
polymer with HPMAm-monolactate39 and eqs 6 and 8 for the
polymer with HPMAm-dilactate): (9) lactoyl lactate; (b) lactic
acid.
k1
k1
[L]t ) [P-LL]0 2 - 2e-(k +k )t
-
×
1
2
{
[
k1 + k2 k3 - k1
k2
k2
co-HPMAm-dilactate)-b-PEG 5000 had a Mn ) 10 800
for the thermosensitive block, and the NIPAAm/HP-
MAm-dilactate molar ratio was 73/27 (mol/mol). The CP
of the block copolymers with the HPMAm-monolactate
was 33.9 °C, while the CP of the block copolymers with
the HPMAm-dilactate was 19.6 °C; after hydrolysis the
CP was 44 °C.
Hydrolysis of the lactic acid side chains was followed
by the formation of lactic acid and lactoyl lactate.
Degradation of the polymers was done at temperatures
between 4 and 60 °C (below and above the cloud point
of the polymers) and at pH 10. Degradation of the poly-
(NIPAAm-co-HPMAm-monolactate) resulted in the for-
mation of lactic acid (L) as degradation product, while
during the degradation of poly(NIPAAm-co-HPMAm-
dilactate) both L and LL (lactoyl lactate) were detected
by HPLC. Typical degradation profiles for the polymers
are shown in Figure 6.
(e-k t - e-k t) - 2
(e-k t - e-k t
)
(8)
1
3
2
4
}
]
[
]
k1 + k2 k4 - k2
All variables in eqs 6 and 8 are known except for k1 and
k2, which can be obtained by the curve fitting procedure
as discussed earlier in this study. As can be seen in
Figure 6, the fitted curves correspond very well to the
experimental data. The Arrhenius plots for the polymers
undergoing the reactions of Scheme 1 are shown in
Figure 7. Ea for the polymer with HPMAm-monolactate
below the cloud point (CP1 ) 31.5 °C, Figure 7) is 95 (
3 kJ /mol, and this value corresponds well with the
activation energy of the hydrolysis of the monomer. No
accurate values for the degradation reactions repre-
sented by k1 and k2 (Scheme 1) for the polymer with
HPMAm-dilactate can be given because k values at only
two temperatures below the CP (CP2, Figure 7) are
available. Discontinuities in the Arrhenius plots (Figure
7) are clearly present around the cloud points of the
polymers. Above the CP, Ea is 37 ( 13 kJ /mol for the
three subreactions.
The same degradation scheme, presented for the
degradation of the monomers, can be applied for deg-
radation of the polymers (Scheme 1, where R is the
polymer backbone).
Table 2 shows the values of the reaction rate con-
stants at pH 7.5 for poly(NIPAAm-co-HPMAm-dilactate)
at 15 °C (below the CP) and 60 °C (above the CP). The
reaction rate constants of the monomers under the same
conditions are also given. The reaction rate constants
for the polymer below its CP are of the same order of
magnitude as those for the monomer, but above the CP
the polymer degrades 1-2 orders of magnitude slower.
The influence of pH on the degradation of the poly-
(NIPAAm-co-HPMAm-mono(di)lactate) polymers was
studied at 60 °C and at pH 8-10. The log k-pH profile
shows that the degradation of poly(NIPAAm-co-
HPMAm-mono(di)lactate) is OH- catalyzed in this pH
range, since the slope of the curve approaches +1
(results not shown).
Knowing the values of the rate constants k3 (0.052
min-1, Figure 6A) and k4 (see monomer degradation
section), the measured amounts of L and LL formed
during the degradation of poly(NIPAAm-co-HPMAm-
dilactate) were fitted using the following equations (eq
6 and 7):
k2
k2
2
4
[LL]t )
[P-LL]
(e-k t - e-k t
) (6)
k1 + k2
0 k4 - k2
and
[L]t ) 2[P-LL]0 - 2[P-LL]t - [P-L]t - 2[LL]t (7)

7496 Neradovic et al.
Macromolecules, Vol. 36, No. 20, 2003
Ta ble 2. Rea ction Ra te Con sta n ts for th e Hyd r olysis of
HP MAm -Dila cta te a n d
P oly(NIP AAm -co-HP MAm -d ila cta te) a t p H 7.5
temp
(°C)
reaction rate
HPMAm-
dilactateb
poly(NIPAAm-co-
HPMAm-dilactate)
constanta (min-1
)
15
k1
k2
k3
k4
k1
k2
k3
k4
4.8 × 10-5
2.4 × 10-5
1.0 × 10-5
1.9 × 10-6
1.3 × 10-2
5.3 × 10-3
3.7 × 10-3
4.7 × 10-4
7.3 × 10-5
4.7 × 10-5
8.4 × 10-6
1.9 × 10-6
9.9 × 10-5
3.8 × 10-4
1.6 × 10-4
4.7 × 10-4
60
a
k1, k2, k3, and k4 refer to the reactions of Scheme 1. b Calculated
reaction rate constants in water using the data of HPMAm-
dilactate degradation in water/DMSO mixture (the presence of 10%
DMSO in the degradation mixture decreased the degradation rate
constants by a factor 2 (Figure 4)).
F igu r e 8. Incubation of poly(NIPAAm-co-HPMAm-dilactate)-
b-PEG 5000 copolymer in 0.15 M NaHCO3 buffer pH 10 and
at 37 °C followed by SLS (9, intensity) and DLS (O, average
particle size (Zave)).
The degradation rate constants for the hydrolysis of
HPMAm-lactate in the poly(NIPAAm-co-HPMAm-mon-
o(di)lactate)-b-PEG 5000 diblock copolymers at 60 °C
and pH 10 corresponded with those for random poly-
(NIPAAm-co-HPMAm-mono(di)lactate) copolymers: deg-
radation rate constants were k1 ) 0.032 min-1, k2 ) 0.20
min-1, and k3 ) 0.056 min-1 (compare with k1 ) 0.031
min-1, k2 ) 0.12 min-1, and k3 ) 0.052 min-1 for the
random polymers).
Since the blocks in the block copolymers are connected
via a hydrolyzable ester bond, the hydrolysis rate of this
bond was also determined. It was shown that, at pH
8.5 and at 37 °C, kobs for the hydrolysis of this ester bond
is (3.4 ( 0.3) × 10-4 min-1 (t1/2 ) 34 h). This means
that the hydrolysis of the ester bond between the blocks
proceeds almost 5 times slower as compared to the
degradation rates of the lactic acid side groups.
F igu r e 9. Formation of HPMAm units during degradation
of poly(NIPAAm-co-HPMAm-dilactate)-b-PEG 5000 at 37 °C
and pH 10, calculated according to eq 5.
P r ed ictive Va lu e of th e Kin etic Mod el. To dem-
onstrate the predictive value of the kinetic model, the
destabilization of nanoparticles based on poly(NIPAAm-
co-HPMAm-dilactate (65/35 (mol/mol)))-b-PEG 5000 at
37 °C and pH 5 and 10 was followed by dynamic and
static light scattering measurements. The experimen-
tally obtained destabilization time was compared with
the calculated time as predicted by the kinetic model.
Dynamic light scattering (DLS) at 37 °C and pH 5, i.e.,
under the conditions where hydrolysis of the polymer
proceeds very slowly, showed that after 5 min incuba-
tion particles of 170 ( 5 nm and with a low polydisper-
sity (PD ) 0.087 ( 0.004) were detected. During the
next 60-70 min of incubation, the particles decreased
in size to 152 ( 4 nm (PD ) 0.085 ( 0.005), which is
likely due to a further dehydration of the hydrophobic
core. After 24 h of incubation, nanoparticles with
approximately the same particle size and polydispersity
(159 ( 3 nm and PD ) 0.09 ( 0.01) were detected. This
demonstrates that under conditions where degradation
is minimized the particles are rather stable.
Destabilization of nanoparticles based on poly-
(NIPAAm-co-HPMAm-dilactate)-b-PEG 5000 was fol-
lowed at 37 °C and pH 10 by DLS and SLS (Figure 8).
Several stages in the destabilization process can be
distinguished. After 5 min of incubation, particles of 190
( 2 nm with a narrow size distribution (PD ) 0.083 (
0.003) were observed in the opalescent polymer solution.
During the next 10 min, both a slight decrease in the
intensity of scattered light and a small but significant
decrease in the particle size to 178 ( 3 nm were
observed. Obviously, because of ongoing dehydration of
the hydrophobic core of the nanoparticles, they become
smaller, as observed for the nanoparticles incubated
under conditions where degradation of the polymer is
minimal (pH 5). During the next 15 min of incubation,
both an increase of the intensity of the scattered light
and the particle size (to 189 ( 3 nm, PD ) 0.08 ( 0.02)
were observed. Likely, because of hydrolysis of the lactic
acid side groups and the herewith associated increase
in hydrophilicity of the polymer, the particles start to
swell, resulting in an increasing particle size in time.
The next phase (30-50 min) is characterized by a
significant increase of the particle size and polydisper-
sity as well as a gradual disappearance of the SLS
signal. This substantial increase in particle size is due
to the increasing hydrophilicity of the hydrophobic
particle core caused by ongoing hydrolysis of the lactic
acid ester groups. After 60 min, the polymer solution
had a clear appearance. In agreement herewith, the SLS
signal was low. These results indicate that between 50
and 60 min the destabilization of the polymeric nano-
particles was complete.
The rate of formation of the HPMAm units during the
polymer degradation can be calculated using eq 5 and
the experimentally determined reaction rate constants
for hydrolysis of the polymer containing di- and mono-
lactic acid side groups. [M]t in eq 5 is equal to the
amount of fully hydrolyzed HPMAm units, and [M-LL]0
is the amount of the polymer bound dilactate at t ) 0,
while [M-LL]t and [M-L]t are the amounts of di- or
monolactic acid still bound to the polymer at time t,
calculated using eqs 4 and 3, respectively.
Figure 9 shows the calculated amount of HPMAm
units during incubation at 37 °C and pH 10. It can be

Macromolecules, Vol. 36, No. 20, 2003
Thermosensitive Polyacrylamides 7497
seen that it takes about 1 h to hydrolyze about 90% of
the side groups from the polymer. As demonstrated
previously,20 the block copolymer formed after hydroly-
sis (poly(NIPAAm-co-HPMAm 65/35) has a cloud point
above 37 °C. This indicates that the kinetic model
predicts that a soluble polymer is formed after 60 min.
SLS and DLS measurements reveal that experimentally
this conversion is found around 50-60 min, which is in
excellent agreement with the calculated destabilization
time.
of the polymer with the monolactate and dilactate side
groups shows first-order kinetics in this temperature
range. From Table 2 it can be seen that at 60 °C the k2
and k3 for the polymer are around ∼10-20 times
smaller than those for the monomer, while k1 for the
polymer is ∼100 times smaller than for the monomer.
This can be explained by fact that, once the polymer is
in its precipitated state, the hydrolyzable ester groups
are in a relatively hydrophobic microenvironment. In
this environment of low dielectric constant the hydroly-
sis slows down (Figure 4). Table 2 also shows that at
60 °C the fastest reaction step for the hydrolysis of the
lactic acid side groups of the polymer is the one
described by k2 (“backbiting”, Scheme 1). This is in
contrast to the degradation of the polymer in the soluble
state, where the degradation of the ester bond between
the two lactate groups is the fastest reaction, as also
observed for the monomer. Obviously, the terminal
hydroxyl group in the precipitated polymer is still
sufficiently mobile to participate in the backbiting
reaction, resulting in the cleavage of the lactoyl lactate.
When the polymer is in a soluble state, k3 < k1 (as for
the monomer). However, in the precipitated state k3 >
k1. This can be explained by the fact that poly(NIPAAm-
co-HPMAm-monolactate) has a higher CP than poly-
(NIPAAm-co-HPMAm-dilactate) and is therefore more
hydrophilic in the precipitated state. It should be noted
that k1 for the polymer at 60 °C is about the same as k1
at 15 °C (the monomer shows an increase of a factor
270). This is a unique observation that despite an
increase of 45 °C the reaction rate does not increase.
Discu ssion
From the overall reaction rate constant (kobs) values
for HPMAm-mono(di)lactate, it is obvious that the
monomer with two lactate groups hydrolyzes 6 times
faster than the monomer with one lactate (compare k1
plus k2 with k3; Table 1). We previously demonstrated
that, during the degradation of a monodisperse lactic
acid oligomer with a blocked carboxylic acid end group,
preferentially lactoyl lactate was split off, which can be
explained by intramolecular transesterification, also
known as backbiting.36 For HPMAm-dilactate the fol-
lowing order in the values of the constants was ob-
tained: k1 > k2 > k3 (Table 1). This indicates that in
HPMAm-dilactate the different ester groups are not
equally susceptible to hydrolysis. The ester group
between the two lactate groups (k1) is more sensitive
for hydrolysis than the one between HPMAm and
lactate (k3). Nevertheless, the “backbiting” mechanism
(k2) still plays a role in the degradation of HPMAm-
dilactate because the HPMAm-lactate ester bond in the
dilactate (k2) is hydrolyzed somewhat faster than the
same ester bond in the monolactate (k3).
The influence of the dielectric constant of the solvent
on the monomer hydrolysis rates was determined for
the following reason. The (NIPAAm-co-HPMAm-lactate)
polymers are soluble in aqueous solutions below their
cloud point, but above this temperature they become
hydrophobic and precipitate. By this process the local
environment of the precipitated polymers becomes
hydrophobic and consequently has a lower dielectric
constant. From Figure 4 it can be seen that, at a higher
dielectric constant of the medium, the degradation
proceeds faster. This is consistent with findings for the
hydrolysis of (poly)esters36-38 and can be explained by
the fact that the organic solvent molecules stabilize the
ground state more than the transition state, resulting
in higher activation energy.
In contrast to the hydrolysis kinetics of the monomers,
the Arrhenius plots for the hydrolysis of HPMAm-
lactate based polymers (Figure 7) clearly show discon-
tinuities around the CP of the polymer. Three separate
regions can be distinguished, i.e., below the CP of the
starting polymer, above the CP of the fully hydrolyzed
polymer, and at intermediate temperatures. The deg-
radation rate constants of the polymers below their CP’s
are almost equal to those for the monomers (Table 2).
Below the cloud point, the polymer is completely dis-
solved and the polymer chains are fully hydrated. In
this state the lactate ester groups are relatively easily
accessible for hydrolysis. As for the monomers, the
degradation of the ester bond between the two lactate
groups (k1) is the fastest reaction, followed by k2 > k3.
When the incubation temperature is in between the
CP’s of the starting polymer and the fully hydrolyzed
polymer (i.e., from 19 to 40 °C for the polymer with
dilactate), then the polymer goes from a precipitated
state to a soluble state during hydrolysis. In contrast
to the observations of Shah et al.,40 who reported a
change in the degradation kinetics for poly(NIPAAm-
co-N-acryloxysuccinimide) which went from a precipi-
tated to a soluble state due to hydrolysis of the side
groups, we found that the polymer degradation followed
first-order kinetics.
Above the CP, the (apparent) activation energies for
hydrolysis of the ester bond between two lactate groups
and the ester bond between the precipitated polymer
backbone and lactate are 37 ( 13 kJ /mol. These very
low (apparent) activation energies for these two reac-
tions can be explained as follows. Above the CP’s two
factors counteract each other. First, a higher tempera-
ture will result in a higher reaction rate. Second, with
increasing temperature, the polymer becomes stronger
dehydrated which is associated with a decrease in
dielectric constant of the microenvironment of the
precipitated polymer. As demonstrated in this paper,
the reaction rate decreases with dielectric constant
(Figure 4). These counteracting effects result in reaction
rate constants that are hardly dependent on tempera-
ture and consequently in a low apparent Ea.
Degradation of block copolymers poly(NIPAAm-co-
HPMAm-mono(di)lactate)/PEG 5000 at 60 °C and at pH
10 results in the same degradation profiles and com-
parable values of the rate constants k1, k2, and k3 with
those obtained for the polymers without the PEG block.
It can thus be concluded that the degradation behavior
of the block copolymers follows the degradation kinetics
and mechanism already discussed for poly(NIPAAm-co-
HPMAm-lactate). Therefore, knowing the degradation
In the temperature range 42-60 °C, the starting
polymer and the polymer obtained after degradation are
both above their CP’s and therefore in the precipitated
state throughout the degradation process. Degradation

7498 Neradovic et al.
Macromolecules, Vol. 36, No. 20, 2003
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ity of the polymeric micelles based on poly(NIPAAm-
co-HPMAm-mono(di)lactate)/PEG under physiological
conditions. Since the degradation rate of the ester bond
between the blocks at 37 °C is almost 5 times slower
compared to the degradation rates of the ester bonds
in the side chains, these micelles will destabilize upon
hydrolysis of the lactic acid side groups due to formation
of more hydrophilic poly(NIPAAm-co-HPMAm)/PEG as
the main degradation product. According to the results
presented in Figures 8 and 9, the kinetic model can very
well predict the destabilization of polymeric nanopar-
ticles. The ability to predict the stability of the men-
tioned polymeric micelles is essential for application of
these systems for drug delivery purposes.
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Con clu sion s
This study shows that, with the in this paper pre-
sented degradation pathways and kinetics, it is possible
to predict and control the destabilization time of poly-
meric micelles based on poly(NIPAAm-co-HPMAm-
lactate)/PEG polymers under physiological conditions.
This is an important feature once the systems are
applied for as drug delivery systems.
Ack n ow led gm en t. The authors thank Dr. W. J . M.
Underberg from the Department of Pharmaceutical
Analysis, Faculty of Pharmaceutical Sciences, Utrecht
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MA034381N