Novel biodegradable cationic core shell nanoparticles for codelivery of drug and DNA 4

68


Chapter 4
Results and Discussion

4.1 Polymer Synthesis
4.1.1 Synthesis and characterization of N-(2-bromoethyl) carbarmoyl cholesterol
(Be-chol)
N-(2-bromoethyl) carbarmoyl cholesterol (Be-chol) has a bromoethyl group that was
used to quaternize the main chain at the amino group and produce positive charges at the
same sites. Be-chol was also designed as the random dispersed hydrophobic pendant
chains. It was synthesized by connecting bromoethylamine onto the cholesterol molecule
through the amidation reaction with cholesteryl chloroformate as showed in Scheme 1.
Be-chol was obtained in yield of ~ 78% after twice consecutive-purification by re-
crystallization with ethanol and acetone, respectively. TLC analysis showed one point at
R
f
of 0.68 in the mixture of toluene, hexane and methanol (8:8:1), indicating that Be-chol
was pure. Figure 4.1 and Figure 4.2 display the
1
H-NMR and FTIR spectra of purified
Be-chol, respectively. As showed in Figure 4.1, the
1
H peak at δ 5.10 (Signal H
N
) was
due to the amide groups (CONH) (See Fig. 4.1). δ 3.60 (Signal H
4
) and 3.61 (Signal H

5
)
were attributed to the 2-bromoethyl groups. δ 4.52 (H
1
) and 5.40 (H
2
) were associated
with the cholesterol units. The ratio of the H
1
,

H
2
,

H
N
, H
4
and H
5
peak areas was
determined to be 1:1:1:2:2, confirming the successful synthesis of Be-chol. The FTIR
spectrum of Be-chol also evidenced its successful synthesis. The IR peak at 3325 cm
-1


69

was due to -NH- stretching (see Figure 4.2). Peaks from -C=O stretching and -NH-

bending overlapped at 1685 cm
-1
. The peak at 1536 cm
-1
was attributed to -C-N-
stretching. In summary, pure Be-chol was successfully synthesized. The purity of Be-chol
is important for its further grafting onto the main chain. For instance, the impurity,
cholesteryl formic acid, may act as a catalyst to promote the hydrolysis of the main chain
(PMDS or PMDA).

Figure 4.1
1
H-NMR spectrum of N-(2-bromoethyl) carbarmoyl cholesterol (Be-chol).
NH
Br
O
H
H
H
O
CH
3
CH
3
H
3
C
H
3
C

H
3
C
a
b
c
d
e f

70


Figure 4.2 FTIR spectrum of N-(2-bromoethyl) carbarmoyl cholesterol (Be-chol).

4.1.2 Synthesis and characterization of PMDS and PMDA
Poly(N-methyldiethylamine sebacate) (PMDS) and Poly(N-methyldiethylamine
adipate) (PMDA) are the main chains of the designed polymers. The successful synthesis
of PMDS was verified by
1
H-NMR and FTIR spectra as shown in Figures 4.3 and Figure
4.4, respectively. NMR peaks at δ 2.71–2.73 (signal a), δ 1.62 (signal b) and δ 1.32
(signals c and d) were attributed to the protons of four different -CH
2
- groups from the
sebacate units (see Figures 4.3). Peaks at δ 4.17–4.19 (signal e) and δ 2.30-2.37 (signals f
and g) were due to protons of two different -CH
2
- groups and the -CH
3
group linked to

the nitrogen atom. IR spectrum also confirmed the polyester formation (see Figure 4.4).
The -C=O stretching shifted to a lower wavenumber (1736 cm
-1
) compared to carbonyl
halide (1805 cm
-1
) due to the inductive effect of halide. The peak at 1172 cm
-1
was
attributed to C-O.

71

The
1
H-NMR and FTIR spectra of PMDA confirmed its successful synthesis (Figure
4.5 and Figure 4.6). NMR peak at δ 1.63 (signal b) was attributed to the -CH
2
- groups of
adipate, which were connected to another two -CH
2
- groups. Peaks at δ 2.67-2.76 (signal
a) were attributed to the -CH
2
- groups of adipate (Figure 4.5), which were connected to
carboxyl group and another -CH
2
- group. Peaks at δ 2.27-2.37 (signals d and e) came
from the protons of the -CH
2

- and -CH
3
groups connected with the nitrogen. Peaks at δ
4.1-4.2 (signal c) were attributed to the -CH
2
- groups connected to the oxygen. The FTIR
spectrum of PMDA also evidenced its successful synthesis (Figure 4.6). Stretching
vibration of C-O at 1174 cm
-1
and stretching vibration of C=O at 1732 cm
-1
were
observed, indicating the existence of ester group in the polymer.
From the
1
H-NMR spectra of PMDS and PMDA, no impurity peaks were observed,
especially the peaks of triethylamine that may influence the subsequent reaction. In
addition, it indicates that the purification method applied was suitable and effective.

Figure 4.3
1
H-NMR spectrum of PMDS.

n
O
N
OC
O
C
O

a
b
c
d
e
f
g
CH
3

72




Figure 4.4 FTIR spectrum of PMDS.

Figure 4.5
1
H-NMR spectrum of PMDA.
n
O
N
O
C
O
C
O
a
b

c
CH
3
d
e

73



Figure 4.6 FTIR spectrum of PMDA.

4.1.3 Synthesis and characterization of P(MDS-co-CES) and P(MDA-co-CEA)
The synthesis of P(MDS-co-CES) and P(MDA-co-CEA) was performed by grafting
Be-chol onto PMDS and PMDA through quaternization reaction. This reaction needs to
be performed at a relatively high temperature when alkyl bromide is used as the reagent
for quaternization. The purposes to introduce the cholesteryl group onto PMDS and
PMDA are to use the cholesteryl group as the core-forming block and to produce positive
charges on the main chain. The successful synthesis of P(MDS-co-CES) and P(MDA-co-
CEA) was evidenced by
1
H-NMR and FTIR spectra. Figure 4.7 and Figure 4.8 show the
1
H-NMR and FTIR spectra of P(MDS-co-CES), respectively. The
1
H-NMR spectrum of
P(MDS-co-CES) illustrates peaks at δ 2.7–2.8 (signal a), 1.5–1.7 (signal b), 1.2–1.4
(signals c and d), 4.0–4.2 (signal e) and 2.2–2.4 (signals f and g) due to protons from the
PMDS main chain (Figure 4.7). Various peaks at δ 0.7–1.2 were attributed to the
cholesterol groups. The peak at δ 5.38 arose from the proton of =CH- in the cholesterol

74

groups (signal h). The peak at δ 0.7 was from the methyl group directly linked to the
cyclic hydrocarbon (signal i). The information provided by the
1
H-NMR spectrum of
P(MDS-co-CES) proved that the cholesteryl group was successfully grafted onto the
PMDS main chain. Figure 4.8 shows the FTIR spectrum of P(MDS-co-CES), which also
evidenced the successful quaternization. The peak at 1252 cm
-1
was attributed to C-N
stretching of amine. The shift and increased intensity of this peak compared with that of
PMDS (1240 cm
-1
) illustrated the formation of a quaternary ammonium salt.
The
1
H-NMR and FTIR spectra of P(MDA-co-CEA) also gave similar results as
P(MDS-co-CES). As shown in Figure 4.9, the wide peak at δ 2.66 (signal a) and the peak
at δ 1.67 (signal b) were from the protons of the methylene groups (-CH
2
-) in the adipate
segments. The multiple peaks at δ 4.0-4.2 (signal c) came from the methylene group (-
CH
2
-) in the N-methyldiethanolamine segments. Signal d of another methylene group
linked to the nitrogen atom of N-methyldiethanolamine was overlapped with signal e of
the methyl group directly linked to the nitrogen at δ 2.3-2.4. The peaks at δ 0.7-1.2 were
from the cholesterol group. In particular, the peaks at δ 5.37 (signal f) and δ 0.69 (signal

g) came from the protons linked to the carbon with double bond (=CH-) and the methyl
group linked to the cyclic hydrocarbon, respectively. Moreover, Figure 4.10 illustrates
the peak at 1251 cm
-1
from the C-N stretching of amine. The shift and increased intensity
of this peak compared with that of PMDA (1240 cm
-1
) illustrated the formation of a
quaternary ammonium salt.
The degree of grafting (R
g
), defined as the ratio of the amines quaternized by N-(2-
bromoethyl) carbarmoyl cholesterol to the total number of amines on the PMDS main
chain, can be estimated as follows,

75

R
g
= (∆A
p
N
Hm
/∆A
m
N
Hp
) × 100%,
Where ∆A
p

is the area of the selected peak from the pendant chain, ∆A
m
is the area of the
selected peak from the main chain, N
Hp
is the number of hydrogen atoms in the selected
group from the pendant chain, and N
Hm
is the number of hydrogen atoms in the selected
group from the main chain. Only suitable protons from the pendant chain and the main
chain of the polymers were selected in the calculation. The proton signal selected should
not overlap with signals from other protons. Furthermore, those protons affected by the
quaternized amines should not be used. For P(MDS-co-CES), the proton of the methylene
group linked to the carbonyl group of the main chain (signal a), as well as the proton of
the methylidyne group (-CH=) linked to the double bond (signal h) and the proton of the
methyl group linked to the hexane and pentane cycles of the pendant chain (signal i) were
considered suitable for use in the estimation of R
g
. For the proton of methylene linked to
the carbonyl group overlapped with other signals. The proton of methylene linked to the
ester (O=C-O-CH
2
-, signal c) on the main chain was chosen since this proton is far from
the nitrogen atom of the quaternary ammonium. The inductive effect of the quaternary
ammonium on the proton was neglected. For the pendant chain, the same protons were
used as P(MDS-co-CES) (i.e. signal f and signal g). Based on the peak areas of signal a
and signal i, R
g
for P(MDS-co-CES) (Batch No. 120902b) was estimated to be 27.0%
(i.e. R

g
=∆A
Hi
×4×100%/3×∆A
Ha
=2.046×4×100%/3×10.1=27.0%). Based on the peak
areas of signal c and signal g, R
g
for P(MDA-co-CEA) (Batch No. 110102b) was
estimated to be 56.0% (i.e. R
g
=∆A
Hg
×4×100%/3×∆A
Hc
=2.59×4×100%/6.17×3=56.0%).
By changing the molar ratio of the pendant chain to the PMDS or PMDA main chain, R
g


76

of the cholesterol moiety and the positive charge of P(MDS-co-CES) could be
modulated.
The polymers with different cholesteryl grafting degree were synthesized by changing
the amount of Be-chol precursor. The purity of PMDS and PMDA may influence the
grafting degree of Be-chol onto PMDS and PMDA. For example, the residue of
triethylamine added to absorb HCl could undergo quaternization reaction with Be-chol.
Therefore, even a small amount of triethylamine and its salt form can affect the grafting
reaction significantly.

Figure 4.7
1
H-NMR spectrum of P(MDS-co-CES).
O
N
p
O
C
O
H
3
C
H
3
C
H
3
C
CH
3
CH
3
O
H
H
H
O
NH
CH

3
Br
+
C
O
O
N
CH
3
q
O
C
O
C
O
a
b
c
d
e
f
g
h
i

77


Figure 4.8 FTIR spectrum of P(MDS-co-CES).

Figure 4.9
1
H-NMR spectrum of P(MDA-co-CEA).
O
N
p
O
C
O
H
3
C
H
3
C
H
3
C
CH
3
CH
3
O
H
H
H
O
NH

CH
3
Br
+
C
O
O
N
CH
3
q
O
C
O
C
O
a
b
c
d
e
f
g

78


Figure 4.10 FTIR spectrum of P(MDA-co-CEA)

4.1.4 PEGylation of PMDS, PMDA and P(MDS-co-CES), P(MDA-co-CEA)

PEGylation means conjugation of PEG onto the polymer. The main purpose of
introducing PEG onto the cationic polymer is to increase the stability of micelles/DNA
complexes by preventing protein absorption. In this study, PEG with molecular weight
(Mn) of 5000, 2000, 1000 and 550 (labeled as PEG5000, PEG2000, PEG1100 and
PEG550 respectively) was conjugated onto PMDS and P(MDS-co-CES) as a terminal
group of the polymers.
Figure 4.11 to Figure 4.15 show the
1
H-NMR spectra of PEG550, PEG1100,
PEG2000, and PEG5000-conjugated PMDS and PEG5000-conjugated PMDA,
respectively. The peak at δ 3.65 was from PEG. An increased molecular weight of PEG
led to increased intensity of the peak, indicating the increase in the relative length of PEG
of the polymer. Figure 4.16 to Figure 4.20 display the
1
H-NMR spectra of PEG550,

79

PEG1100, PEG2000, and PEG5000-conjugated P(MDS-co-CES) and PEG5000-
conjugated P(MDA-co-CEA), respectively. Similarly, the intensity of the peak at δ 3.65
increased as increasing the molecular weight of PEG, indicating the increase in the
relative length of PEG of the polymer. Peaks at δ 0.7-1.2 were from the cholesteryl
groups. The single peak at δ 0.69 was attributed to the methyl group linked to the cyclic
hydrocarbon. These results show that the grafting of cholesteryl group and the
conjugation of PEG onto PMDS and PMDA were successful. However, the grafting
degree of cholesteryl group onto PEGylated PMDS and PMDA was much lower than that
onto PMDS and PMDA (Table 4.1, and Figure 4.16-4.20). That means that PEG
influenced the quaternization reaction of Be-chol with the tertiary amine on the PMDS
and PMDA main chains. It is possibly because the conjugation of PEG onto PMDS and
PMDA changed the polarity of the polymer and the compatibility of the polymer with

toluene, which restricted fully stretching of the polymer chain in toluene and thus provide
steric hindrance to the quaternization reaction.

Figure 4.11
1
H-NMR spectrum of PEG550-PMDS.

80


Figure 4.12
1
H-NMR spectrum of PEG1100-PMDS.

Figure 4.13
1
H-NMR spectrum of PEG2000-PMDS.

Figure 4.14
1
H-NMR spectrum of PEG5000-PMDS.

81



.
Figure 4.15
1
H-NMR spectrum of PEG5000-PMDA.

Figure 4.16
1
H-NMR spectrum of PEG550-P(MDS-co-CES).




82




Figure 4.17
1
H-NMR spectrum of PEG1100-P(MDS-co-CES).


Figure 4.18
1
H-NMR spectrum of PEG2000-P(MDS-co-CES).





83

Figure 4.19
1
H-NMR spectrum of PEG5000-P(MDS-co-CES).

Figure 4.20
1
H-NMR spectrum of PEG5000-P(MDA-co-CEA).

4.1.5 Molecular weight, grafting degree as well as PEG contents of the polymers
The molecular weight, grafting degree and PEG contents of the polymers are listed in
Table 4.1. The batch numbers of the polymers was named after the synthesis date. The
grafting degree was obtained from the
1
H-NMR spectra. The PEG content was estimated

84

from the
1
H-NMR spectra. The weight average molecular weight (Mw) of PMDS could
reach as high as 18.5 kDa while the Mw of P(MDS-co-CES) could be up to 7.9 kDa. The
molecular weight of P(MDS-co-CES) was usually lower than the PMDS, from which the
P(MDS-co-CES) was synthesized. This indicates that the grafting reaction may result in
the degradation of the main chain, decreasing the molecular weight. The cholesteryl
grafting degree of P(MDS-co-CES) ranged from 9.4% to 56.2%, depending on the purity
of PMDS and the amount of Be-chol added. As discussed in the previous sections,
triethylamine in PMDS could significantly affect the grafting degree. The
1
H-NMR
spectra of PMDS evidenced that extracting PMDS/toluene solution using NaCl-saturated

aqueous solution for at least 4 times effectively removed the triethylamine and thus
increased the grafting degree when other conditions remained the same. The amount of
Be-chol used in the quaternization reaction also influenced the grafting degree. For
example, polymers with the batch numbers of 191003a and 191003b were synthesized by
using Be-chol to PMDS unit ratios of 0.5 and 0.2 respectively. The grafting degree
obtained were 13.6% and 9.4% respectively (Table 4.1). However, the grafting degree
seldom exceeded 60% even though the molar ratio of Be-chol to PMDS unit increased to
1.5 (Batch No.010902). This is possibly because the structure of the cholesteryl group
provided steric hindrance for the reaction. For PEGylated P(MDS-co-CES) polymers, the
grafting degrees ranged from 4.25% to 16.35%, where the molar ratio of Be-chol to
PMDS unit was at 1.0 except for PEG2000-P(MDS-co-CES) (Batch No. 051103b), for
which the ratio of 0.5 was used. Obviously, the grafting degrees of PEGylated P(MDS-
co-CES) were much lower than that of P(MDS-co-CES). As discussed in Section 4.1.4,
PEG-conjugated PMDS may influence the conformation of the chain in the solvent and

85

thus hinder the grafting reaction. This is possibly the main factor that renders the low
grafting degree of PEG-P(MDS-co-CES). In in vitro gene expression experiments these
polymers, with or without grafted PEG or cholesteryl groups, will be tested to uncover
the influence of polymer structure on gene transfection level.

4.1.6 Thermal properties of the polymers
The thermal properties of polymeric materials are a key factor that determines their
storage conditions and sterilization methods to be employed. The thermal properties of
PMDA, P(MDA-co-CEA), PMDS and P(MDS-co-CES) polymers were studied using
TGA and DSC. Figure 4.21 to Figure 4.24 show the TGA spectra of PMDA and P(MDA-
co-CEA), PMDS and P(MDS-co-CES), respectively. The maximal degradation
temperature of P(MDS-co-CES) and P(MDA-co-CEA) appeared at 339.7ºC and 339.8ºC,
respectively while those of PMDS and PMDA occurred at temperatures higher than

400ºC. The similar thermal degradation patterns between P(MDS-co-CES) and P(MDA-
co-CEA) as well as between PMDS and PMDA may be due to their similar chemical
structure. The lower degradation temperature of P(MDS-co-CES) and P(MDA-co-CEA)
compared with PMDS and PMDA suggests that the quaternization of the tertiary amine
on the main chain may make the polymer more degradable. The dissociation of
cholesteryl groups from the main chain may also cause the degradation to occur at lower
temperatures. From the cumulative degradation curves of these polymers, there was very
small weight loss starting from 100ºC, which was water loss. The small water loss shows
that the moisture existing in the polymers was not high. This may be due to the

86

hydrophobic nature of the polymers. This indicates that the storage and transportation
conditions should not be stringent.
From Figure 4.25 and Figure 4.26, it can be seen that the glass transition temperature
of P(MDS-co-CES) and P(MDA-co-CEA) was 73.5ºC and 24.3ºC, respectively. The
melting point of P(MDS-co-CES) was also observed at 138.2ºC.

Figure 4.21 TGA spectrum of PMDA (Batch No. 110902).

Figure 4.22 TGA spectrum of P(MDA-co-CEA) (Batch No. 111002a).


87


Figure 4.23 TGA spectrum of PMDS (Batch No. 120902a).

Figure 4.24 TGA spectrum of P(MDS-co-CES) (Batch No. 120902b).

Figure 4.25 DSC spectrum of P(MDS-co-CES) (Batch No. 120902b).

88


Figure 4.26 DSC spectrum of P(MDA-co-CEA) (Batch No. 111002a).

4.1.7 In vitro degradation of P(MDS-co-CES) and P(MDA-co-CEA)
The main chains of P(MDS-co-CES) and P(MDA-co-CEA) are polyester. It is known
that polyester is degradable in aqueous solution, especially in an acidic environment.
Degradable materials are easily accepted by scientists in the biomedical field since the
degradable materials can be cleared out of the body through the renal system during the
degrading process and prevent the accumulation of the foreign materials in the body. The
in vitro degradation tests of P(MDS-co-CES) and P(MDA-co-CEA) were performed in
PBS (pH 7.4). Figure 4.27 shows the weight loss of the polymers as function of
incubation time. On the third day, the weight of P(MDS-co-CES) slightly increased
because of water uptake and ions from the PBS buffer. After that, it underwent rapid
weight loss. At Week 8, its weight loss was about 54%. P(MDA-co-CEA) experienced
much slower degradation. After eight weeks incubation, only 25% weight loss was found.
The samples harvested after the weight loss test can neither dissolve in water nor organic
solvents including DMF, which makes the measurement of molecular weight of the

89

degraded polymers difficult using GPC. This also makes the characterization of the
residue by
1
H-NMR and FTIR very difficult. The weight loss of P(MDS-co-CES) after
the third day is mainly due to the hydrolysis of the main PMDS chain. Both sebacic acid
and methyl diethanolamine produced after hydrolysis were soluble in PBS. Thus, the

main residue of the polymers after eight weeks of degradation should be cholesteryl
group, which was not soluble in PBS. Similarly, P(MDA-co-CEA) had a great amount of
residue (75%) after eight weeks of degradation. Because the grafting degree of P(MDA-
co-CEA) used was higher than that of P(MDS-co-CES) (56.0% versus 27.0%) and the
unit molecular weight of PMDA is also lower than that of PMDS, the weight loss of
P(MDA-co-CEA) was slower than that of P(MDS-co-CES). However, the figure 4.27
also shows that P(MDA-co-CEA) can be hydrolyzed more easily, since , unlike P(MDS-
co-CES), the weight of PMDA-co-CEA didn’t increase at the third day.
In summary, the weight loss of polymer in the period of 8 weeks can surely evidence the
degradation of the polymer. Although the difficulty of detecting degraded product due to
the ionization of the polymer make the study of degradation mechanism impossible, it is
reasonable to say that the polymer should be degraded by hydrolysis since it possess ester
structure.

90

0
20
40
60
80
100
120
0 10 20 30 40 50 60
Time (days)
%original weight of samples
PMDS-co-CES PMDA-co-CEA

Figure 4.27 Degradation of P(MDS-co-CES) (Batch No. 120902b) and P(MDA-co-CEA)
(Batch No. 111002b).

Table 4.1 Molecular weight, grafting degree and PEG content of the polymers.
Batch
No.
Polymer Name Mw Mn Mw/Mn MW
PEG
:W
M
PEG-PMDS

Grafting
Degree (%)
260802 PMDS 15100 9700 1.5 / /
010902 P(MDS-co-CES) (260802)* 9100 4500 2.0 / 56.2
110902 PMDA 3500 2800 1.3 / /
111002a P(MDA-co-CEA) (110902)* 3000 2700 1.1 / 56.0
120902b P(MDS-co-CES) 5800 3200 1.8 / 27.0
151002 P(MDS-co-CES) 3300 2500 1.3 / 33.0
201002 P(MDS-co-CES) 3200 2400 1.3 / 46.7
300603 PMDA 4900 3200 1.5 / /
120803 PMDA 5100 3200 1.6 / /
310803 PMDS 7300 3700 2.0 / /
191003a P(MDS-co-CES) (310803)* 4800 3400 1.4 / 13.6
191003b P(MDS-co-CES) (310803)* 4600 2900 1.6 / 9.4

91

301003 PMDS 12500 5800 2.1 / /
110204a P(MDS-co-CES) (301003) 5400 2900 1.8 30.25
110204b P(MDA-co-CEA) (120803)* 1700 1600 1.0 / 34.4

040304 PMDS 11200 4800 2.3 / /
260304 P(MDS-co-CES) (040304)* 7900 3300 2.4 / 26.5%
010704 P(MDS-co-CES) 5000 3000 1.7 / 28.5%
081003 PEG550-PMDS 6000 3400 1.8 6.2% /
171103 PEG1100-PMDS 9500 3800 2.5 13% /
101003 PEG2000-PMDS 6400 3500 1.8 20% /
140703b PEG5000-PMDS 23500 13100 1.8 / /
140703a PEG5000-PMDA 13400 11500 1.2 / /
011203 PEG550-P(MDS-co-CES)
(081003)*
13400 8500 1.6 1.8% 12.15
281203 PEG1100-P(MDS-co-CES)
(171103)*
15800 8900 1.8 7.5% 4.25
051103A PEG2000-P(MDS-co-CES)
(101003)*
3900 2600 1.5 7.6% 16.35
051103B PEG2000-P(MDS-co-CES)
(101003)*
5100 3300 1.5 8.2% 8.62
310703b PEG5000-P(MDS-co-CES)
(140703b)*
12800 8500 1.5 / 2.05
310703a PEG5000-co-P(MDA-co-CEA)
(140703a)*
11400 8900 1.3 / 1.78
* Batch No. of the precursor polymer

4.2 Fabrication and characterization of polymeric micelles
4.2.1 Micelles formation and CMC determination

92

The critical micelle concentration (CMC) is an important factor to evaluate the
micelles formation by amphiphilic polymers through self-assembly [Jones M-C., 1999].
In addition, it is also a critical factor to understand the stability of micelles in the post
blood administration.
Pyrene is a commonly used fluorescent probe to measure CMC of amphiphilic
polymers. With the formation of micelles, hydrophobic pyrene goes into the hydrophobic
core of micelles. In this procedure the microenvironment of pyrene changes from
hydrophilic to hydrophobic, the ratio of I
1
/I
3
from the emission spectra of pyrene
decreases but the ratio of I
338
/I
333
from its excitation spectra increases abruptly near the
CMC. Since the I
1
/I
3
ratio is affected by the excitation wavelength and may result in an
erroneous CMC value [Jones M-C., 1999], both emission and excitation spectra were
used to ascertain the formation of micelles. Since P(MDS-co-CES) is a polyelectrolyte,
the effect of pH and ionic strength on the CMC of P(MDS-co-CES) was also studied.
Figure 4.28 and Figure 4.29 show the CMC of P(MDS-co-CES) in deionized water and
the sodium acetate buffer with different pH and ionic strength, measured by the changes

of I
1
/I
3
and I
338
/I
333
as a function of polymer concentration. The CMC measured from the
emission spectra was 6.1 mg/L in deionized water and 7.4 mg/L in the sodium acetate
buffer (0.1M, pH 4.6) (see Figure 4.28). However, from the excitation spectra, it was 1.9
mg/L and 2.4 mg/L respectively. The CMC of P(MDS-co-CES) in the sodium acetate
buffer (0.02M, pH 4.6) was 5.2 mg/L and 1.5 mg/L, obtained from the emission and
excitation spectra respectively. The CMC of P(MDS-co-CES) in the sodium acetate
buffer (0.02M, pH 5.6) was 6.0 mg/L and 1.9 mg/L, measured from the emission and
excitation spectra respectively. The real CMC of the polymer should be between the two