The anionic derivative of asymmetric triblock copolymer containing biocompatible chemically complementary
polyacrylamide and poly(ethylene oxide) (PAAm-b-PEO-b-PAAm) was obtained. The micellization
of the initial (TBC) and modified (TBC-COOH) copolymer samples in aqueous solution were investigated.
Practically the same values of the critical micellization concentration and the Gibbs free micellization energy
for both TBCs were found. The anticancer effects of the doxorubicin (DOX)-loaded micelles of the initial
and modified triblock copolymers were studied on the tumor cells of human T-leukemia of Jurkat line,
transformed leukemia of L929 line and mouse lymphatic leukemia of L1210 line. The fact of high efficacy of
DOX/TBC and DOX/TBC-COOH compositions in compare with pure DOX was determined and discussed.
When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/3497

Boiko, N.M

Kunitskaya, L.R.

Permyakova, N.M.

Shlyakhtina, Ye.A.

Stoika, R.S.

Zheltonozhskaya, T.B.

SocioEconomic Challenges Journal (Sumy State University)

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE NANOMATERIALS: APPLICATIONS AND PROPERTIES 
Vol. 1 No 2, 02NNBM10(3pp) (2012) 
 
 
2304-1862/2012/1(2)02NNBM10(3) 02NNBM10-1  2012 Sumy State University 
Anionic Micelle-Forming Triblock Copolymers as Nanocontainers for Doxorubicin 
 
L.R. Kunitskaya1,*, T.B. Zheltonozhskaya1,†, N.M. Permyakova1,  
R.S. Stoika2, N.M. Boiko2, Ye.A. Shlyakhtina2  
 
1 Kiev National Taras Shevchenko University, 60 Vladimirskaya St, 01033 Kiev, Ukraine 
2 Institute of Cell Biology, NAS Ukraine, 14/15 Dragomanova St., 79005 Lvov, Ukraine 
 
(Received 31 May 2012; revised manuscript received 04 July 2012; published online13 July 2012) 
 
The anionic derivative of asymmetric triblock copolymer containing biocompatible chemically comple-
mentary polyacrylamide and poly(ethylene oxide) (PAAm-b-PEO-b-PAAm) was obtained. The micellization 
of the initial (TBC) and modified (TBC-COOH) copolymer samples in aqueous solution were investigated. 
Practically the same values of the critical micellization concentration and the Gibbs free micellization en-
ergy for both TBCs were found. The anticancer effects of the doxorubicin (DOX)-loaded micelles of the ini-
tial and modified triblock copolymers were studied on the tumor cells of human T-leukemia of Jurkat line, 
transformed leukemia of L929 line and mouse lymphatic leukemia of L1210 line. The fact of high efficacy of 
DOX/TBC and DOX/TBC-COOH compositions in compare with pure DOX was determined and discussed. 
 
Keywords: Amphiphilic Block Copolymers, Drug, Micellization, Poly(Ethylene Oxide), Polyacrylamide. 
 
 PACS number: 62.23.St 
 
 
                                                          
* larisa_kunitskaya@ukr.net 
† zheltonozhskaya@ukr.net 
1. INTRODUCTION 
 
Amphiphilic block copolymers are widely studied for 
pharmaceutical application. Polymeric micelles self-
assembled from amphiphilic block copolymers have 
been intensively investigated as nano-carrier systems 
for tumor-targeted drug delivery of toxic and poorly 
soluble drugs to achieve improved cancer chemothera-
py[1-3]. The continuous development of new drug carri-
ers is driven by the need to maximize therapeutic activ-
ity while minimizing negative side effects. It was 
shown earlier [4, 5] that asymmetric diblock (DBC) and 
triblock (TBC) copolymers contained biocompatible 
chemically complementary polyacrylamide and 
poly(ethylene oxide) (PAAm-b-PEO-b-PAAm) or its 
monomethyl ether (MOPEO-b-PAAm) formed special 
micellar structures in aqueous solutions (Figure 1). 
 
 
Fig. 1 – Scheme of micelle formation 
 
Such structures contain hydrophobic “core” formed 
by hydrogen-bonded PEO and PAAm chains and hy-
drophilic “corona” of the surplus segments of PAAm. 
Essential influence of the anticancer agent doxorubicin 
(DOX) on the micellization process due to the interac-
tion between DOX and copolymer micelles was estab-
lished. This opened new prospects for using such copol-
ymers as carriers for DOX and other toxic drugs.  
The present study examines the possible use of anionic 
derivatives of PAAm-b-PEO-b-PAAm as potential carries 
for DOX. Major attention is paid to the micelle formation 
of modified TBC in aqueous solutions and in vitro study of 
antitumor effect of the DOX-loaded modified TBC mi-
celles as compared to the initial one and free DOX. 
2. EXPERIMENTAL PART 
 
2.1 Materials and Methods 
 
The triblock copolymer was synthesized by the radi-
cal block copolymerization of PAAm with PEG initiated 
by CeIV ions according to the method described earlier 
[5]. The sample of PEG with Mn  6 kDa and cerium 
ammonium nitrate (initiator) from Aldrich (USA) were 
used for this purpose. The molecular weights of PAAm 
blocks and TBC macromolecules (MnPAAm  117 kDa, 
MnTBC  240 kDa) were determined from 1H NMR spec-
tra recorded in D2O at C  1 kg m – 3 and a room tem-
perature using a Varian Mercury-400 spectrometer 
operating at 400 MHz. The structure of TBC was con-
formed by FTIR spectrum measured by a Nexus-470 
Nicolet (USA) spectrometer with a resolution 4 cm – 1. 
The alkaline hydrolysis reaction was performed in 
accordance with the following scheme: 
 
 
 
Aqueous solutions of TBC and individual PAAm 
(Mv  3500 kDа) with C  10 kg m – 3 were stirred at the 
presence of NaOH (CNaOH  5 mol∙l – 1) at Т  50 С. Sam-
ple splitting was realized via 10, 60, 75, 240 and 480 
minutes. The samples of anionic derivatives were modi-
fied into H-form by the acidification up to Рн  2. Gel-
like TBCs were re-precipitated by acetone, dissolved in 
water and freeze-dried. Kinetic investigations of the al-
kaline hydrolysis process were performed by potentiom-
etric titration. The initial and modified TBC samples 
were titrated as compared to H2O using 2N NaOH.  
The critical micellization concentration (CMC) of 
TBCs was determined by static light scattering (SLS) 
using a modernized light scattering instrument FRS-3 
(Russia) contained WP7113VGC/A light-emitting diode 
(   520 nm) from “Kingbright”, ADC-CPUTM controller 
from “Insoftus” (Ukraine) and the computer program 
_ (- CH
2
_
CH_
CONH
2
) _
n +
mNaOH
_ (_ CH
n
)
2
CONH
_CH_
2
_m
__ )_CH
_
2
CH-(
m
+ mNH
3
COO
_
Na
+
 L.R. KUNITSKAYA, T.B. ZHELTONOZHSKAYA, N.M. PERMYAKOVA, ET AL. PROC. NAP 1, 02NNBM10 (2012) 
 
 
02NNBM10-2 
WINRECODER. In order to define CMCs, the scatter-
ing intensities of the vertically polarized light were 
measured at the   90  scattering angle and T  20 C. 
Biological studies in vitro were performed using 
three cellular models: murine leukemia of L1210 line, 
transformed leukemia of L929 line and acute T-cell hu-
man leukemia of Jukat line. The L1210 and L929 cells 
were incubated in DMEM medium, Jurkat cells in 
RPMI medium, in the presence of 10 % bovine serum. 
Incubation was performed in 96-hole plates (“Falcon”, 
USA) in the CO2 incubator (“JENCONS NUAIRE”, UK). 
To compare the action of free DOX, DOX/nanocarrier 
complex and free nanocarrier, the concentrations of 
compounds were calculated according to the concentra-
tion of DOX. The half-lethal dose (LD50) of DOX for 
mentioned above cell lines is 1 μg/cm3. The effectiveness 
of the investigated substances was determined accord-
ing to the number of alive cells relative to their number 
in control (the number of cells in culture medium with-
out addition of the drug). The counting of cell number 
was carried out in hemocytometer chamber in 24 and 48 
hours after incubation with drugs. Trypan blue was 
used for counting dead cells. 
 
3. RESULTS AND DISCUSSION 
 
The results of TBC hydrolysis as compare to PAAm 
is shown in Table 1.  
 
Table 1 – Parameters of TBC and PAAm alkaline hydrolysis 
 
Copolymer t, 1) 
min 
σlim∙103, 2) 
g-ekv∙g – 1 
А, 3) 
% 
Vh·104, 
4)s – 1 
TBC-
COOH 
0 
10 
60 
75 
240 
480 
0.18 
1.51 
2.85 
3.15 
4.82 
5.39 
1.4 
10.7 
20.3 
22.4 
34.4 
38.5 
1.6 
PAAm 10 
240 
0.29 
0.66 
10.2 
23.4 
0.4 
1) The hydrolysis time. 2) The limit value of hydroxyl ion absorp-
tion. 3) The hydrolysis degree. 4) The initial rate of hydrolysis. 
 
It is seen that the alkaline hydrolysis process devel-
oped with higher rate in TBC than in pure PAAm. 
The only modified TBC sample (TBC-COOH) be-
longing to 10 min of hydrolysis time was chosen for our 
further experiments and tested by potentiometric titra-
tion. The titration curve for the modified TBC was used 
to calculate the hydroxyl ion absorption [7 8]. As it fol-
lows from Figure 2, the TBC-COOH absorption curve 
has S-shaped character. The limit value of the hydroxyl 
ion absorption ( lim) corresponded to the quantity of 
carboxyl groups in the polymer sample, which was 
achieved at pH  8.5-9.0.  
 
 
Fig. 2 – Curves of (a) OH− ion absorbtion and (b) pK depend-
ence from the ionization degree of –COOH groups in TBC-
COOH (A  10,7 %). CTBC−COOH  2 kg m – 3; Т  25 С 
On the basis of Figure 2 a, the hydrolysis degree was 
calculated according to the (3.1) equation: 
 
 
3
lim
3
lim
10
100%
'
10
71
PAAm
A
w
 (3.1) 
 
where w PAAm is the weight fraction of PAAm chains in 
the modified TBC-COOH sample. The dependence of 
negative logarithm of the apparent dissociation con-
stant (pK) versus the dissociation degree ( ) was calcu-
lated from Figure 2 a by an ordinary relation (3.2): 
 
 lg
1
pK pH  (3.2) 
 
Here   c/ lim, where c is the current hydroxyl ion 
absorption at a certain pH. As we can see from Figure 2 
b, TBC-COOH demonstrates the properties of a weak 
polyelectrolyte without any conformation transition 
during its ionization. 
In the movement of using the initial and modified 
TBCs as nanocarriers for target delivery of toxic and 
poorly soluble drugs, it was important to examine the 
micelle formation of mentioned block copolymers in 
water solution. Micellization occurs in dilute solutions 
of block copolymers at a fixed temperature starting 
from some concentration called the critical micelliza-
tion concentration (CMC) [5]. The CMCs of TBC and 
TBC-COOH were determined by SLS. Using CMC and 
thermodynamic theory of micellization the Gibbs free 
micellization energy was calculated as: 
 
 lnoG RT CMC , (3.3) 
 
Both the parameters are submitted in Table 2 and 
show practically the same CMC and - G  values for 
initial and modified TBC samples. Thereby, such insig-
nificant substitution of amide groups with carboxyl 
ones in PAAm blocks of TBC does not lead to additional 
micelle stabilization in aqueous medium. 
 
Table 2 – Micelle formation characteristics 
  
Copolymer CMC  108, 1) 
mol  dm – 3 
- G ,2) 
kJ mol – 1 
TBC 
TBC-COOH 
37.8 
39.8 
36.15 
36.14 
1) The critical micellization concentration. 2) The Gibbs free 
micellization energy  
 
In order to estimate the binding ability of the initial 
and modified TBCs in respect of toxic hydrophobic 
drugs, the anticancer activity one of the most effective 
antitumor agent DOX was studied in the presence of 
the mentioned block copolymers. DOX molecule (Figure 
3) has sufficiently developed hydrophobic part and also 
active hydroxyl-, ether-, carbonyl- and amine groups. It 
is reasonable to suppose that – OH groups of DOX are 
able of hydrogen-bonding interaction with ether groups 
of PEO block and amide groups of PAAm blocks of TBC. 
Moreover, we also relied to improve encapsulation of 
DOX in result of strong electrostatic interactions be-
tween amine and carboxyl groups via introduction of 
COOH-groups into TBC macromolecule by means of 
alkaline hydrolysis of PAAm blocks.  
 ANIONIC MICELLE-FORMING TRIBLOCK … PROC. NAP 1, 02NNBM10 (2012) 
 
 
02NNBM10-3 
 
 
Fig. 3 – Molecular structure of doxorubicin 
 
The anticancer activity of DOX encapsulated by mi-
celles of the initial and modified TBCs was investigated 
in vitro against murine leukemia of L1210 line, trans-
formed leukemia of L929 line and acute T-cell human 
leukemia of Jukat line (Figure 4-6). 
 
 
 
Fig. 4 – Dynamics of living cells of T-cell human leukemia in the 
presence of DOX-loaded TBC –2 and TBC-COOH –3 micelles 
compared to initial DOX –1. The incubation time is 24 h 
 
a  
b  
Fig. 5 – Dynamics of living cells of mouse lymphatic leukemia 
in the presence of DOX-loaded TBC – 2 and TBC-COOH – 3 
micelles as compared to pure DOX – 1. The incubation time is 
24 h (a) and 48 h (b) 
 
 
Fig. 6 – Dynamics of living cells of transformed leukemia in 
the presence of DOX-loaded TBC – 2 and TBC-COOH – 3 mi-
celles unlike to pure DOX – 1. The incubation time is 24 h 
 
The obtained results suggest that DOX/TBC-COOH 
composition significantly enhance the antitumor effect 
of doxorubicin in the range of concentrations 0.10-
1.00 µg ml – 1 especially through 48 h (the example is 
shown in Figure 5 b). The anticancer activity of this 
composition is higher than that of DOX/TBC composi-
tion and pure DOX practically in all cultural mediums 
under study. It is connected with increase in the bind-
ing capability of the modified P(AAm-co-AAc) blocks of 
TBC with respect to DOX molecules due to strong elec-
trostatic interactions of amine and carboxylic groups. 
 
4. CONCLUSION 
 
The anionic derivative of PAAm-b-PEO-b-PAAm 
triblock copolymer with relatively small hydrolysis de-
gree forms micellar structures because of intramolecu-
lar complex formation of P(AAm-co-AAc) and PEO 
blocks. They are characterized practically the same sta-
bility in aqueous medium as the micelles of non-
modified TBC sample that was confirmed by close CMC 
and  – ∆G  values. 
The composition DOX/TBC-COOH express essen-
tially stronger anticancer activity than DOX/TBC com-
position and pure DOX against all the tumor lines test-
ed. At the same time, the DOX/TBC composition turns 
out to be more effective than pure DOX at the long-
term incubation (48 h). Optimization of doses and study 
of the mechanism of DOX/TBC-COOH action is under 
way. However, the experimental results can be useful 
for further development of efficient nanovehicles for 
delivery of cytotoxic drugs.  
 
 
 
REFERENCES 
 
1. Y. Chan, T. Wong, F. Byrne, M. Kavalaris, V. Bulmus, 
Biomacromolecules 9,1826 (2008) 
2. K.T. Oh, E. S. Lee, Polym Adv. Tech. 19, 1907 (2008). 
3.  Y. Wang, V. Bansal, A. Zelikin, F. Caruso, Nano Letter 8, 
1741 (2008) 
4. L.R. Kunitskaya, et al. Mol. Cryst. Liq. Cryst. 536, 398 
(2011) 
5. T.B. Zheltonozhskaya at al. Mol. Cryst. Liq. Cryst. 536, 
390 (2011) 
6. G. Riess, Prog. Polym. Sci. 28, 1107 (2005). 
7. T. Zheltonozhskaya, Vysokomol. Soed. 23, 2425 (1981).
 


English

Anionic Micelle-Forming Triblock Copolymers as Nanocontainers for Doxorubicin

Electronic Sumy State University Institutional Repository

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE NANOMATERIALS: APPLICATIONS AND PROPERTIES Vol. 1 No 2, 02NNBM10(3pp) (2012)   2304-1862/2012/1(2)02NNBM10(3) 02NNBM10-1  2012 Sumy State University Anionic Micelle-Forming Triblock Copolymers as Nanocontainers for Doxorubicin  L.R. Kunitskaya1,*, T.B. Zheltonozhskaya1,†, N.M. Permyakova1,  R.S. Stoika2, N.M. Boiko2, Ye.A. Shlyakhtina2   1 Kiev National Taras Shevchenko University, 60 Vladimirskaya St, 01033 Kiev, Ukraine 2 Institute of Cell Biology, NAS Ukraine, 14/15 Dragomanova St., 79005 Lvov, Ukraine  (Received 31 May 2012; revised manuscript received 04 July 2012; published online13 July 2012)  The anionic derivative of asymmetric triblock copolymer containing biocompatible chemically comple-mentary polyacrylamide and poly(ethylene oxide) (PAAm-b-PEO-b-PAAm) was obtained. The micellization of the initial (TBC) and modified (TBC-COOH) copolymer samples in aqueous solution were investigated. Practically the same values of the critical micellization concentration and the Gibbs free micellization en-ergy for both TBCs were found. The anticancer effects of the doxorubicin (DOX)-loaded micelles of the ini-tial and modified triblock copolymers were studied on the tumor cells of human T-leukemia of Jurkat line, transformed leukemia of L929 line and mouse lymphatic leukemia of L1210 line. The fact of high efficacy of DOX/TBC and DOX/TBC-COOH compositions in compare with pure DOX was determined and discussed.  Keywords: Amphiphilic Block Copolymers, Drug, Micellization, Poly(Ethylene Oxide), Polyacrylamide.   PACS number: 62.23.St                                                             * larisa_kunitskaya@ukr.net † zheltonozhskaya@ukr.net 1. INTRODUCTION  Amphiphilic block copolymers are widely studied for pharmaceutical application. Polymeric micelles self-assembled from amphiphilic block copolymers have been intensively investigated as nano-carrier systems for tumor-targeted drug delivery of toxic and poorly soluble drugs to achieve improved cancer chemothera-py[1-3]. The continuous development of new drug carri-ers is driven by the need to maximize therapeutic activ-ity while minimizing negative side effects. It was shown earlier [4, 5] that asymmetric diblock (DBC) and triblock (TBC) copolymers contained biocompatible chemically complementary polyacrylamide and poly(ethylene oxide) (PAAm-b-PEO-b-PAAm) or its monomethyl ether (MOPEO-b-PAAm) formed special micellar structures in aqueous solutions (Figure 1).   Fig. 1 – Scheme of micelle formation  Such structures contain hydrophobic “core” formed by hydrogen-bonded PEO and PAAm chains and hy-drophilic “corona” of the surplus segments of PAAm. Essential influence of the anticancer agent doxorubicin (DOX) on the micellization process due to the interac-tion between DOX and copolymer micelles was estab-lished. This opened new prospects for using such copol-ymers as carriers for DOX and other toxic drugs.  The present study examines the possible use of anionic derivatives of PAAm-b-PEO-b-PAAm as potential carries for DOX. Major attention is paid to the micelle formation of modified TBC in aqueous solutions and in vitro study of antitumor effect of the DOX-loaded modified TBC mi-celles as compared to the initial one and free DOX. 2. EXPERIMENTAL PART  2.1 Materials and Methods  The triblock copolymer was synthesized by the radi-cal block copolymerization of PAAm with PEG initiated by CeIV ions according to the method described earlier [5]. The sample of PEG with Mn  6 kDa and cerium ammonium nitrate (initiator) from Aldrich (USA) were used for this purpose. The molecular weights of PAAm blocks and TBC macromolecules (MnPAAm  117 kDa, MnTBC  240 kDa) were determined from 1H NMR spec-tra recorded in D2O at C  1 kg m – 3 and a room tem-perature using a Varian Mercury-400 spectrometer operating at 400 MHz. The structure of TBC was con-formed by FTIR spectrum measured by a Nexus-470 Nicolet (USA) spectrometer with a resolution 4 cm – 1. The alkaline hydrolysis reaction was performed in accordance with the following scheme:    Aqueous solutions of TBC and individual PAAm (Mv  3500 kDа) with C  10 kg m – 3 were stirred at the presence of NaOH (CNaOH  5 mol∙l – 1) at Т  50 С. Sam-ple splitting was realized via 10, 60, 75, 240 and 480 minutes. The samples of anionic derivatives were modi-fied into H-form by the acidification up to Рн  2. Gel-like TBCs were re-precipitated by acetone, dissolved in water and freeze-dried. Kinetic investigations of the al-kaline hydrolysis process were performed by potentiom-etric titration. The initial and modified TBC samples were titrated as compared to H2O using 2N NaOH.  The critical micellization concentration (CMC) of TBCs was determined by static light scattering (SLS) using a modernized light scattering instrument FRS-3 (Russia) contained WP7113VGC/A light-emitting diode (   520 nm) from “Kingbright”, ADC-CPUTM controller from “Insoftus” (Ukraine) and the computer program _ (- CH2_CH_CONH2) _n +mNaOH_ (_ CHn)2CONH_CH_2_m__ )_CH_2CH-(m+ mNH3COO_Na+ L.R. KUNITSKAYA, T.B. ZHELTONOZHSKAYA, N.M. PERMYAKOVA, ET AL. PROC. NAP 1, 02NNBM10 (2012)   02NNBM10-2 WINRECODER. In order to define CMCs, the scatter-ing intensities of the vertically polarized light were measured at the   90  scattering angle and T  20 C. Biological studies in vitro were performed using three cellular models: murine leukemia of L1210 line, transformed leukemia of L929 line and acute T-cell hu-man leukemia of Jukat line. The L1210 and L929 cells were incubated in DMEM medium, Jurkat cells in RPMI medium, in the presence of 10 % bovine serum. Incubation was performed in 96-hole plates (“Falcon”, USA) in the CO2 incubator (“JENCONS NUAIRE”, UK). To compare the action of free DOX, DOX/nanocarrier complex and free nanocarrier, the concentrations of compounds were calculated according to the concentra-tion of DOX. The half-lethal dose (LD50) of DOX for mentioned above cell lines is 1 μg/cm3. The effectiveness of the investigated substances was determined accord-ing to the number of alive cells relative to their number in control (the number of cells in culture medium with-out addition of the drug). The counting of cell number was carried out in hemocytometer chamber in 24 and 48 hours after incubation with drugs. Trypan blue was used for counting dead cells.  3. RESULTS AND DISCUSSION  The results of TBC hydrolysis as compare to PAAm is shown in Table 1.   Table 1 – Parameters of TBC and PAAm alkaline hydrolysis  Copolymer t, 1) min σlim∙103, 2) g-ekv∙g – 1 А, 3) % Vh·104, 4)s – 1 TBC-COOH 0 10 60 75 240 480 0.18 1.51 2.85 3.15 4.82 5.39 1.4 10.7 20.3 22.4 34.4 38.5 1.6 PAAm 10 240 0.29 0.66 10.2 23.4 0.4 1) The hydrolysis time. 2) The limit value of hydroxyl ion absorp-tion. 3) The hydrolysis degree. 4) The initial rate of hydrolysis.  It is seen that the alkaline hydrolysis process devel-oped with higher rate in TBC than in pure PAAm. The only modified TBC sample (TBC-COOH) be-longing to 10 min of hydrolysis time was chosen for our further experiments and tested by potentiometric titra-tion. The titration curve for the modified TBC was used to calculate the hydroxyl ion absorption [7 8]. As it fol-lows from Figure 2, the TBC-COOH absorption curve has S-shaped character. The limit value of the hydroxyl ion absorption ( lim) corresponded to the quantity of carboxyl groups in the polymer sample, which was achieved at pH  8.5-9.0.    Fig. 2 – Curves of (a) OH− ion absorbtion and (b) pK depend-ence from the ionization degree of –COOH groups in TBC-COOH (A  10,7 %). CTBC−COOH  2 kg m – 3; Т  25 С On the basis of Figure 2 a, the hydrolysis degree was calculated according to the (3.1) equation:   3lim3lim10100%'1071PAAmAw (3.1)  where w PAAm is the weight fraction of PAAm chains in the modified TBC-COOH sample. The dependence of negative logarithm of the apparent dissociation con-stant (pK) versus the dissociation degree ( ) was calcu-lated from Figure 2 a by an ordinary relation (3.2):   lg1pK pH  (3.2)  Here   c/ lim, where c is the current hydroxyl ion absorption at a certain pH. As we can see from Figure 2 b, TBC-COOH demonstrates the properties of a weak polyelectrolyte without any conformation transition during its ionization. In the movement of using the initial and modified TBCs as nanocarriers for target delivery of toxic and poorly soluble drugs, it was important to examine the micelle formation of mentioned block copolymers in water solution. Micellization occurs in dilute solutions of block copolymers at a fixed temperature starting from some concentration called the critical micelliza-tion concentration (CMC) [5]. The CMCs of TBC and TBC-COOH were determined by SLS. Using CMC and thermodynamic theory of micellization the Gibbs free micellization energy was calculated as:   lnoG RT CMC , (3.3)  Both the parameters are submitted in Table 2 and show practically the same CMC and - G  values for initial and modified TBC samples. Thereby, such insig-nificant substitution of amide groups with carboxyl ones in PAAm blocks of TBC does not lead to additional micelle stabilization in aqueous medium.  Table 2 – Micelle formation characteristics   Copolymer CMC  108, 1) mol  dm – 3 - G ,2) kJ mol – 1 TBC TBC-COOH 37.8 39.8 36.15 36.14 1) The critical micellization concentration. 2) The Gibbs free micellization energy   In order to estimate the binding ability of the initial and modified TBCs in respect of toxic hydrophobic drugs, the anticancer activity one of the most effective antitumor agent DOX was studied in the presence of the mentioned block copolymers. DOX molecule (Figure 3) has sufficiently developed hydrophobic part and also active hydroxyl-, ether-, carbonyl- and amine groups. It is reasonable to suppose that – OH groups of DOX are able of hydrogen-bonding interaction with ether groups of PEO block and amide groups of PAAm blocks of TBC. Moreover, we also relied to improve encapsulation of DOX in result of strong electrostatic interactions be-tween amine and carboxyl groups via introduction of COOH-groups into TBC macromolecule by means of alkaline hydrolysis of PAAm blocks.   ANIONIC MICELLE-FORMING TRIBLOCK … PROC. NAP 1, 02NNBM10 (2012)   02NNBM10-3   Fig. 3 – Molecular structure of doxorubicin  The anticancer activity of DOX encapsulated by mi-celles of the initial and modified TBCs was investigated in vitro against murine leukemia of L1210 line, trans-formed leukemia of L929 line and acute T-cell human leukemia of Jukat line (Figure 4-6).    Fig. 4 – Dynamics of living cells of T-cell human leukemia in the presence of DOX-loaded TBC –2 and TBC-COOH –3 micelles compared to initial DOX –1. The incubation time is 24 h  a  b  Fig. 5 – Dynamics of living cells of mouse lymphatic leukemia in the presence of DOX-loaded TBC – 2 and TBC-COOH – 3 micelles as compared to pure DOX – 1. The incubation time is 24 h (a) and 48 h (b)   Fig. 6 – Dynamics of living cells of transformed leukemia in the presence of DOX-loaded TBC – 2 and TBC-COOH – 3 mi-celles unlike to pure DOX – 1. The incubation time is 24 h  The obtained results suggest that DOX/TBC-COOH composition significantly enhance the antitumor effect of doxorubicin in the range of concentrations 0.10-1.00 µg ml – 1 especially through 48 h (the example is shown in Figure 5 b). The anticancer activity of this composition is higher than that of DOX/TBC composi-tion and pure DOX practically in all cultural mediums under study. It is connected with increase in the bind-ing capability of the modified P(AAm-co-AAc) blocks of TBC with respect to DOX molecules due to strong elec-trostatic interactions of amine and carboxylic groups.  4. CONCLUSION  The anionic derivative of PAAm-b-PEO-b-PAAm triblock copolymer with relatively small hydrolysis de-gree forms micellar structures because of intramolecu-lar complex formation of P(AAm-co-AAc) and PEO blocks. They are characterized practically the same sta-bility in aqueous medium as the micelles of non-modified TBC sample that was confirmed by close CMC and  – ∆G  values. The composition DOX/TBC-COOH express essen-tially stronger anticancer activity than DOX/TBC com-position and pure DOX against all the tumor lines test-ed. At the same time, the DOX/TBC composition turns out to be more effective than pure DOX at the long-term incubation (48 h). Optimization of doses and study of the mechanism of DOX/TBC-COOH action is under way. However, the experimental results can be useful for further development of efficient nanovehicles for delivery of cytotoxic drugs.     REFERENCES  1. Y. Chan, T. Wong, F. Byrne, M. Kavalaris, V. Bulmus, Biomacromolecules 9,1826 (2008) 2. K.T. Oh, E. S. Lee, Polym Adv. Tech. 19, 1907 (2008). 3.  Y. Wang, V. Bansal, A. Zelikin, F. Caruso, Nano Letter 8, 1741 (2008) 4. L.R. Kunitskaya, et al. Mol. Cryst. Liq. Cryst. 536, 398 (2011) 5. T.B. Zheltonozhskaya at al. Mol. Cryst. Liq. Cryst. 536, 390 (2011) 6. G. Riess, Prog. Polym. Sci. 28, 1107 (2005). 7. T. Zheltonozhskaya, Vysokomol. Soed. 23, 2425 (1981). 

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Anionic Micelle-Forming Triblock Copolymers as Nanocontainers for Doxorubicin

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