Nanostructure formation in aqueous solution of amphiphilic copolymers of 2-(N,N-dimethylaminoethyl)methacrylate and alkylacrylate: Characterization, antimicrobial activity, DNA binding, and cytotoxicity studies

Article abstract of DOI:10.1016/j.ijpharm.2011.05.006

Three amphiphilic random copolymers poly(2-(dimethylaminoethyl) methacrylate-co-alkylacrylate) (where, alkyl = hexyl, octyl, dodecyl) with 16 mol% hydrophobic substitution were synthesized. Surface tension, viscosity, fluorescence probe, dynamic light scattering (DLS), as well as transmission electron microscopic (TEM) techniques were utilized to investigate self-assembly formation by the hydrophobically modified polymers (HMPs) in pH 5. Formation of hydrophobic domains through inter-polymer chain interaction of the copolymer in dilute solution was confirmed by fluorescence probe studies. Average hydrodynamic diameter of the copolymer aggregates at different polymer concentration was measured by DLS studies. The copolymer with shorter hydrophobic chain exhibits larger hydrodynamic diameter in dilute solution, which decreased with either increase of concentration or increase of hydrophobic chain length. TEM images of the dilute solutions of the copolymers with shorter as well as with longer hydrophobic chain exhibit spherical aggregates of different sizes. The antimicrobial activity of the copolymers was evaluated by measuring the minimum inhibitory concentration value against one Gram-positive bacterium Bacillus subtilis and one Gram-negative bacterium Escherichia coli. The copolymer with the octyl group as pendent hydrophobic chain was found to be more effective in killing these microorganisms. The interaction of the cationic copolymers with calf-thymus DNA was studied by fluorescence quenching method. The polymer-DNA binding was found to be purely electrostatic in nature. The hydrophobes on the polymer backbone were found to have a significant influence on the binding process. Biocompatibility studies of the copolymers in terms of cytotoxicity measurements were finally performed at different concentrations of the HMPs to evaluate their potential application in biomedical fields.

Full text of DOI:10.1016/j.ijpharm.2011.05.006

International Journal of Pharmaceutics 414 (2011) 298–311
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical Nanotechnology
Nanostructure formation in aqueous solution of amphiphilic copolymers of
-(N,N-dimethylaminoethyl)methacrylate and alkylacrylate: Characterization,
antimicrobial activity, DNA binding, and cytotoxicity studies
2
Pranabesh Dutta , Joykrishna Dey , Anshupriya Shomeb,1, Prasanta Kumar Das
a
a,∗
b,∗∗
a
Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India
Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Three amphiphilic random copolymers poly(2-(dimethylaminoethyl)methacrylate-co-alkylacrylate)
where, alkyl = hexyl, octyl, dodecyl) with 16 mol% hydrophobic substitution were synthesized. Surface
Received 12 March 2011
Received in revised form 1 May 2011
Accepted 2 May 2011
(
tension, viscosity, fluorescence probe, dynamic light scattering (DLS), as well as transmission electron
microscopic (TEM) techniques were utilized to investigate self-assembly formation by the hydrophobi-
cally modified polymers (HMPs) in pH 5. Formation of hydrophobic domains through inter-polymer chain
interaction of the copolymer in dilute solution was confirmed by fluorescence probe studies. Average
hydrodynamic diameter of the copolymer aggregates at different polymer concentration was measured
by DLS studies. The copolymer with shorter hydrophobic chain exhibits larger hydrodynamic diameter in
dilute solution, which decreased with either increase of concentration or increase of hydrophobic chain
length. TEM images of the dilute solutions of the copolymers with shorter as well as with longer hydropho-
bic chain exhibit spherical aggregates of different sizes. The antimicrobial activity of the copolymers was
evaluated by measuring the minimum inhibitory concentration value against one Gram-positive bac-
terium Bacillus subtilis and one Gram-negative bacterium Escherichia coli. The copolymer with the octyl
group as pendent hydrophobic chain was found to be more effective in killing these microorganisms. The
interaction of the cationic copolymers with calf-thymus DNA was studied by fluorescence quenching
method. The polymer-DNA binding was found to be purely electrostatic in nature. The hydrophobes on
the polymer backbone were found to have a significant influence on the binding process. Biocompatibil-
ity studies of the copolymers in terms of cytotoxicity measurements were finally performed at different
concentrations of the HMPs to evaluate their potential application in biomedical fields.
Available online 11 May 2011
Keywords:
Hydrophobically modified polymer
Transmission electron microscopy
Antimicrobial activity
DNA binding
Biocompatibility
©
2011 Elsevier B.V. All rights reserved.
1
. Introduction
these cationic copolymers is their ability of killing pathogenic
microorganisms and acting as an antimicrobial agent which can
be used in areas such as healthcare products, food storage, water
purification systems, household sanitation, medical devices, etc.
These are mostly nonvolatile, chemically stable, and more effi-
cient in comparison to the commonly used low-molecular-weight
antimicrobial agents (Kawabata, 1992; Tashiro, 2001). As a result,
continuous efforts have been made for the last few years to
develop polycations with antimicrobial function (i.e. polymeric bio-
cides). The mechanism of antimicrobial activity depends largely
on the hydrophilic–hydrophobic balance. Since the long chain
hydrophobic functionalities provide a compatibility with the lipid
bilayer of the bacterial cytoplasmic membrane, it is important to
have optimum/thresholds hydrophobic functionalities for diffu-
sion through the cell membrane (Ikeda et al., 1986; Nonaka et al.,
Water-soluble cationic polymers have been the subject of con-
tinuing investigation over the years. These have received great
attention due to their important role in biomedical applications,
especially as carriers for controlled drug release (Zhang et al.,
2
008), scaffolds for tissue engineering (Malafaya et al., 2007),
and vehicles for gene transfer therapy (Lv et al., 2006; Park
et al., 2006). Various cationic copolymers like poly(l-lysine) (Niwa
et al., 2002), polyethylenimine (PEI) (Lungwitz et al., 2005), and
derivatives of chitosan (Kim et al., 2007), have been synthesized
and studied for the same. One additional attractive feature for
∗
Corresponding author. Tel.: +91 3222 283308; fax: +91 3222 255303.
2
003). Based on this background several hundred to thousands
∗
∗
Corresponding author. Tel.: +91 33 2473 4971; fax: +91 33 2473 2805.
E-mail address: joydey@chem.iitkgp.ernet.in (J. Dey).
Tel.: +91 33 24734971; fax: +91 33 24732805.
of polymeric compounds with varying lipophilic–lipophobic bal-
ances have been synthesized and tested for different microbes
1
0
378-5173/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2011.05.006

P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
299
(
Kenawy et al., 2007; Palermo et al., 2009). However, it has been
found that the antimicrobial activities of polycations having sim-
ilar structures can vary dramatically and there is no such strict
structure–property relationship for the cationic polymers. One
possible reason behind this heterogeneity might be due to the poly-
disperse nature of the polymers with varying range of molecular
weights (Kanazawa et al., 1993). In fact, the average molecular
weight of polymers plays a substantial role in governing the bio-
logical function especially microbial activity, toxicity, etc.Among
various other water-soluble cationic polymers investigated so far,
poly(2-(dimethylaminoethyl)methacrylate, poly(DMAEMA) is one
such polymer that has gained potential application starting from
drug/gene delivery to water purification systems (Van de Wetering
et al., 1997; Gu et al., 2009; Hoogeveen et al., 1996; Zhu et al., 2009;
Keely et al., 2009). This is a weak polyelectrolyte with pKa ≈ 7.9
Fig. 1. Synthetic scheme for the preparation of cationic copolymers reported in this
work.
be required for killing those pathogenic microorganisms. More-
over, the detailed self-assembly studies along with the evaluation
of antimicrobial activity, DNA binding ability, and toxicity will
open up opportunities for their use in other areas also. There-
fore, in this article we have extended the strategy to introduce
hydrophobic modification of DMAEMA based polyelectrolyte with
different hyhdrophobic side chain. Three random copolymers of
DMAEMA and alkylacrylate (where alkyl = hexyl, octyl, dodecyl)
with 16 mol% hydrophobic substitution (Fig. 1) were synthesized
and their self-assembly properties were studied in terms of intra-
and intermolecular interaction in aqueous media combining sur-
face tension, viscometry, fluorescence, dynamic light scattering
(
Merle, 1987; Pradny and Sevcik, 1985) in water, which is close to
the physiological pH and shows lower critical solution temperature,
LCST, behavior quite similar to PNIPAM. Depending on the molec-
ular weight, pH, and salt concentration various values in the range
◦
of 38–50 C have been reported for its LCST in aqueous media (Lee
et al., 2003). In regards to their application as antimicrobial agents
it has been demonstrated that this homopolymer can inhibit the
growth of Escherichia coli and Bacillus subtilis when attached to glass
(
Lee et al., 2004; Huang et al., 2008), filter paper (Lee et al., 2004;
Roy et al., 2008), polystyrene (Lenoir et al., 2005), and polypropy-
lene (Huang et al., 2007). Very recently, Rawlinson et al. (2010) have
carried out a detailed investigation on the antibacterial activity of
unconjugated and 50% conjugated poly(DMAEMA) polymer against
a range of bacterial strain. They have found that poly(DMAEMA)
acts as bacteriostatic against Gram-negative bacteria with MIC
values between 0.1 and 1 mg/mL. They observed that in case of
Gram-negative bacteria it is active around its pKa and at higher
pH values, while for Gram-positive bacteria the poly(DMAEMA) is
active around its pKa and at lower pH values.
(DLS), and transmission electron microscopy (TEM) techniques.
Effects of concentration, pH, electrolyte, and temperature have
been investigated on the aggregation behavior of these hydropho-
bically modified polyelectrolytes (HMPs). Finally, antimicrobial
activity, DNA binding ability, and toxicity measurements were per-
formed to explore the potential applications of these copolymers.
The poly(DMAEMA) homopolymer has also been established as
an efficient gene carrier (Wetering et al., 1998). It binds plasmid
DNA resulting polymer/plasmid complexes (commonly known as
polyplexes) and introduces DNA into cells. The lower pKa value
of poly(DMAEMA) amino groups is believed to contribute to its
high transfection efficiency (Wetering et al., 1999). The trans-
fection efficiency of poly(DMAEMA) can further be modulated
with incorporation of hydrophilic or hydrophobic functional group
into this polymer. Consequently, various hydrophilic/hydrophobic
monomers were polymerized with DMAEMA to investigate their
transfection properties (Tan et al., 2006; Rungsardthong et al.,
2. Experimental
2.1. Materials
The monomer 2-(N, N-dimethylaminoethyl)methacrylate
(DMAEMA) (Aldrich) was purified by reported procedure
(Georgiou et al., 2005). Dodecanol, octanol, hexanol (SRL),
acryloyl chloride, chloroform-d (CDCl3), deuterium oxide (D2O),
and
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) (Aldrich) were used without further purifica-
tion. The radical initiator 2,2 -azobis(isobutyronitrile), AIBN
ꢀ
2
001). The hydrophobic monomers lead to stable complex for-
(Aldrich) was recrystallized from methanol. The fluorescence
probes, such as pyrene, 1,6-diphenyl-1,3,5-hexatriene (DPH), and
N-phenyl-1-naphthylamine (NPN) (Aldrich) were recrystallized
from ethanol or acetone-ethanol mixture three times before use.
Calf-thymus deoxyribonucleic acid (Ct-DNA) (SRL, Mumbai) and
ethidium bromide (SRL, Mumbai) were used as received. Analytical
grade sodium hydroxide, sodium bicarbonate, sodium hydrogen
phosphate, sodium dihydrogen phosphate, sodium chloride and
hydrochloric acid were used directly from the bottle. All the
solvents, ethanol, methanol, tetrahydrofuran (THF), acetone, and
dichloromethane were of commercially available and were dried
and distilled fresh before use. Doubly distilled water was used for
preparation of all solution.
mation with DNA for efficient cell uptake and prevent enzymatic
degradation of DNA. This causes higher transfection efficiency
than polymer systems using only ionic interactions. Kurisawa and
coworkers observed that in the case of water-soluble terpolymer
poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethyl
methacrylate (DMAEMA)-co-butylmethacrylate (BMA)) containing
2
5
0 mol% of DMAEMA, the transfection efficiency with 0, 2, and
mol% of BMA was low. However, when the BMA content increases
to 10 mol%, a 2-fold higher transfection efficiency than that of the
poly(DMAEMA) control homopolymer can be achieved (Kurisawa
et al., 2000).
Although the antimicrobial activity of poly(DMAEMA) has been
investigated either in its unconjugated or quaternized ammo-
nium form, to our knowledge, there is hardly any systematic
report of the microbial activity, toxicity, and self-assembly prop-
erties for this polymer with hydrophobic side chain of different
chain length. Introduction of hydrophobic monomer units onto the
poly(DMAEMA) backbone will create an amphiphilic structure in
which hydrophilic/hydrophobic ratio will govern the biocidal activ-
ity of the copolymers and much less amount of compound will
2.2. Synthesis of monomers
Dodecylacrylate (DA), octylacrylate (OA), and hexylacrylate
(HA) were prepared by acylation of the respective alcohol with
acryloyl chloride in a manner analogous to the synthesis of N-
alkylacrylamide reported by Morishima et al. (Morishima et al.,
1989). Briefly, in a 100 mL round-bottom flask fitted with mag-

3
00
P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
netic stirrer n-alkanol (0.01 mol) was dissolved in 10 mL of THF.
2.3.1. Poly(2-(dimethylaminoethyl)methacrylate-co-
hexylacrylate) [poly(DMAEMA-HA)]
A solution of triethylamine (0.01 mol) in 10 mL of THF was then
◦
−1
added. The reaction mixture was cooled in an ice bath below 10 C.
FT-IR (KBr, cm ): 3411 (N–H stretching), 2932, 2837
−
1
Acryloyl chloride (0.012 mol) was added drop wise into the mix-
ture. After the addition, the solution was stirred with magnetic
stirrer for additional 3 h at room temperature. The reaction mix-
ture was poured into water and extracted with dichloromethane.
The organic layer was washed three times with water and dried
over anhydrous magnesium sulfate. After evaporation of the sol-
vent the residue was purified by column chromatography using
silica gel and was identified by elemental analysis, 1H NMR, and
FT-IR spectroscopy.
(alkyl C–H stretching), 1728 cm
(C O stretch, ester group),
−
1
1
1259 cm (C–N stretching), H NMR (200 MHz, CDCl + CD OD
(20 ␮L)), ( (ı in ppm): 4.31 (br, 2H, –O–CH –CH –NH(CH ) ,
3
3
2
2
3 2
DMAEMA), 2.85 (br, 6H, –O–CH –CH –NH(CH ) , DMAEMA),
2
2
3 2
1.64 (br, 6H, –O–CH –CH –(CH ) –CH , HAc), 0.84 (m, 3H,
2
2
2
3
3
–O–CH –CH –(CH ) –CH , HAc), 1.83 (br, 1H, –CH–CH –, main
2
2
2
3
3
2
chain), 2.54 (br, 2H, –CH–CH –, main chain).
2
2.3.2. Poly(2-(dimethylaminoethyl)methacrylate-
co-octylacrylate) [poly(DMAEMA-OA)]
−
1
FT-IR (KBr, cm ): 3409 (N–H stretching), 2941, 2840
2
.2.1. Dodecylacrylate (DA)
(
−
1
(
1
alkyl C–H stretching), 1734 cm
257 cm
(C O stretch, ester group),
Yield 84%) FT-IR (KBr, cm−1): 2926, 2855 (C–H stretching);
−
1
(C–N stretching), 1H NMR (200 MHz, CDCl + CD OD
3
3
1
1
729 (C O stretch of ester); 1637 (–C C-stretching); 1466, 1407,
(
20 ␮L)), ( (ı in ppm): 4.39 (br, 2H, –O-CH –CH –NH(CH ) ,
271, 1191; 1H NMR (200 MHz, CDCl ), ( (ı in ppm): 6.41 (dd, 1H,
2 2 3 2
3
DMAEMA), 2.92 (br, 6H, –O–CH –CH –NH(CH ) , DMAEMA),
2
2
3 2
H C CH), 6.18 (dd, 1H, HHC CH–), 5.81 (dd, 1H, HHC CH–), 4.21
2
1
–
.72 (br, 6H, –O–CH –CH –(CH )5–CH , OAc), 0.88 (m, 3H,
2 2 2 3
(
t, 2H, O–CH –CH –), 1.67 (m, 2H, O–CH –CH –(CH ) –CH ),
2 2 2 2 2 9 3
O–CH –CH –(CH )5–CH , OAc), 1.94 (br, 1H, –CH–CH –, main
2
2
2
3
2
1
.29 (m, 18H, O–CH –CH –(CH ) –CH ), 0.80 (t, 3H,
2 2 2 9 3
chain), 2.58 (br, 2H, –CH–CH –, main chain).
2
O–CH –CH –(CH ) –CH ); CHN analysis: calcd. (%): C, 74.94,
2
2
2
9
3
H, 11.74; found: C, 74.37, H, 11.17.
2.3.3. Poly(2-(dimethylaminoethyl)methacrylate-co-
dodecylacrylate) [poly(DMAEMA-DA)]
−
1
2
.2.2. Octylacrylate (OA)
(
FT-IR (KBr, cm ): 3408 (N–H stretching), 2937 (alkyl
Yield 79%) FT-IR (KBr, cm−1): 2922, 2852 (C–H stretching);1727
C–H stretching), 1731 cm
−1
(C
O
stretch, ester group),
−
1
(C–N stretching), 1H NMR (200 MHz, CDCl + CD OD
(
C
O stretch of ester); 1630 (–C C-stretching); 1462, 1404, 1263,
1260 cm
(20 ␮L)), ( (ı in ppm): 4.28 (br, 2H, –O–CH –CH –NH(CH ) ,
3
3
1
189; 1H NMR (200 MHz, CDCl ), ( (ı in ppm): 6.35 (dd, 1H,
3
2
2
3 2
H C CH), 6.10 (dd, 1H, HHC CH–), 5.80 (dd, 1H, HHC CH–), 4.14
2
DMAEMA), 2.79 (br, 6H, –O–CH –CH –NH(CH ) , DMAEMA),
2 2 3 2
(
t, 2H, O–CH –CH –), 1.64 (m, 2H, O–CH –CH –(CH )5–CH ),
1.58 (br, 6H, –O–CH –CH –(CH ) –CH , DAc), 0.87 (m, 3H,
2 2 2 9 3
2
2
2
2
2
3
1
.32 (m, 10H, O–CH –CH –(CH )5–CH ), 0.85 (t, 3H,
–O–CH –CH –(CH ) –CH , DAc), 1.87 (br, 1H, –CH–CH –, main
2
2
2
3
2 2 2 9 3 2
O–CH –CH –(CH )5–CH ); CHN analysis calcd. (%): C, 71.69,
chain), 2.51 (br, 2H, –CH–CH –, main chain).
2
2
2
2
3
H, 10.93; found: C, 71.55, H, 11.17.
2.4. General instrumentation
2
.2.3. Hexylacrylate (HA)
(
The 1H NMR spectra were recorded on a Brucker SEM 200 spec-
Yield 81%) FT-IR (KBr, cm−1): 2932 (C–H stretching); 1723 (C
O
trophotometer using CDCl3 as solvent and trimethyl silane (TMS)
as internal standards. For weighing of the samples, semi-micro-
analytical balance (Sartorious, model: CP2225D) were used. The
UV–vis spectra were recorded with a Shimazdu (model: 1601) spec-
trophotometer. The circular dichroism (CD) spectra were recorded
with a Jasco J-810 spectropolarimeter using quartz cell of 10 mm
path length. An average of four scan of each spectrum was taken
under the conditions of 1-nm bandwidth, 2-s response time, and
50 nm/min scan speed. Each spectrum was corrected by sub-
tracting the appropriate reference blank. The pH measurements
were performed with a digital pH meter (pH 5652, EC India Ltd.,
Kolkata) using glass electrode. Average molecular weight and
molecular weight distribution of the copolymers in their depro-
tonated form were obtained by gel permeation chromatography
(GPC) using a Waters GPC instrument equipped with Styragel HR-4
columns. Tetrahydrofuran (THF) was used as an eluent (flow rate
0.6 mL/min). The molecular weight and polydispersity index were
calculated from the calibration curve previously constructed from
monodispersed polystyrene standard.
stretch of ester); 1633 (–C C-stretching); 1458, 1404, 1271, 1190;
1
H NMR (200 MHz, CDCl ), ( (ı in ppm): 6.38 (dd, 1H, H C CH),
3
2
6
.09 (dd, 1H, HHC CH–), 5.79 (dd, 1H, HHC CH–), 4.11 (t, 2H,
O–CH –CH –), 1.63 (m, 2H, O-CH -CH -(CH ) -CH ), 1.34 (m, 6H,
2
2
2
2
2
3
3
O–CH –CH –(CH ) -CH ), 0.87 (t, 3H, O–CH –CH –(CH ) –CH );
2
2
2
3
3
2
2
2
3
3
CHN analysis: calcd. (%): C, 69.19, H, 10.32. Found: C, 68.87, H, 10.56.
2.3. Synthesis of copolymers
The three tertiary amine-based cationic HMPs were prepared by
copolymerization of DMAEMA and alkylacrylate (16 mol%), similar
to the synthetic pathway reported recently by our group (Dutta
et al., 2009). Briefly, DMAEMA and alkylacrylate (HA, OA or DA)
were dissolved in DMF with pre-determined weight ratio. Oxygen-
free nitrogen was purged through the solution mixture for 45 min at
◦
6
0 C. AIBN (1 mol% of the total monomer concentration) dissolved
in DMF was added via syringe. The polymerization was continued
for 24 h. Finally, it was recovered by passing dry HCl gas through
the reaction mixture. For complete precipitation, the mixture was
poured into large excess of chloroform. To purify the copoly-
mers, repeated precipitation from methanol with chloroform was
done and the product was finally dissolved in pure water. Subse-
quently, the solution was dialyzed (MWCO 12,000–14,000 g/mol)
against distilled water (pH 6–7) for 1 week and then lyophilized.
The copolymers were chemically identified by FT-IR and 1H NMR
spectroscopy. The synthetic scheme of polymerization reaction
is presented in Fig. 1. Conveniently, the HMPs with different
hydrophobe chain length (6, 8, and 12) were abbreviated as follows:
poly(DMAEMA-HA), poly(DMAEMA-OA), and poly(DMAEMA-DA).
2.5. Surface tension measurement
To check the surface activity of the polymer, surface tension
(ꢀ) were measured in aqueous buffer solution of copolymer with
a surface tensiometer (model 3S, GBX, France) using Du Nuöy ring
detachment method. Before every measurement the platinum ring
was cleaned properly with ethanol–HCl solution and checked the
surface tension of distilled water. Aliquot of polymer stock solution
made in buffer solution (pH 5) was gradually added to a beaker
containing known volume of the same buffer and stirred gently

P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
301
with a magnet. Before each measurement, the solution was allowed
to stand for about 30 min to equilibrate at 30 C and the mean of
relation curves obtained were analyzed using the CONTIN software
provided by Malvern.
◦
three measurements was taken as equilibrium surface tension.
2.9. Transmission electron microscopy
2.6. Viscosity measurement
The morphology of the copolymer aggregates in solution was
An Ubbelohde glass viscometer (ASTM-D-446) with efflux time
investigated by transmission electron microscopy. The samples
were prepared by immersing a 400 mesh size carbon-coated cop-
per grid in to the copolymer solution (1 g/L) for 1 min. The excess
liquid was blotted off with filter paper, air-dried, and kept in a
moisture-free desiccators prior use. The measurements were car-
ried on a JEOL-JEM 2100 (Japan) electron microscope operating at
an accelerating voltage of 200 kV at room temperature.
of 180 s (for water) was used to measure the viscosity of aque-
ous polymer solutions. The sample as well as the viscometer was
◦
equilibrated at 30 C throughout the experiment. At least five read-
ings were taken to determine the average flow–through time of the
copolymer solution at various concentrations (0.05–3 g/L). Specific
viscosity was determined by comparison with the flow-through
time of buffer alone. For determination of density of the copolymer
solutions, a portable digital density meter (Densito 30 PX, Mettler-
Toledo, GmbH) was used.
2.10. Ethidium bromide displacement assay
The binding abilities of the cationic copolymers to ct-DNA were
investigated by ethidium bromide displacement assay. A 500 ␮L of
polymer solution of desired concentration (0.0005–0.016 g/L) was
2.7. Steady-state fluorescence spectra
The steady-state fluorescence measurements of aqueous poly-
added into equal volumes of ct-DNA/EB solution to make final ct-
−7
−6
mer solution containing pyrene (5.0 × 10 M) were performed on a
DNA concentration of 0.01 g/L and EB concentration of 2.0 × 10
M
−
3
SPEX Fluorolog-3 spectrofluorometer. A stock solution (1 × 10 M)
in methanol was added to the polymer solution of different concen-
tration, gently shaken and allowed to equilibrate for 24 h before
measurement. The solutions were excited at 335 nm and the emis-
sion was recorded in the wavelength range 350–500 nm. The ratio
of intensities of the first (I , at ∼372 nm) and the third (I , at
to obtain polymer/DNA complexes with various weight ratios.
Before measurement, the solutions were equilibrated for 12 h
under dark condition. The final solutions were then excited at
530 nm and the emission was recorded in the wavelength range
500–700 nm.
1
3
∼
383 nm) vibronic peaks of the pyrene fluorescence spectrum
2.11. Antimicrobial assay
was used as a measure of the polarity in the local environment.
Stock solutions of NPN were prepared by adding the compound
to buffer solution and stirring magnetically for 24 h. The excess
compound was removed by filtration through Millipore syringe fil-
ter (0.22 ␮m) and the samples prepared with this stock solution
were excited at 340 nm. The emission was collected in the range
In vitro antimicrobial activities of the cationic HMPs were eval-
uated using measurements of minimum inhibitory concentration
(MIC) against both Gram-positive and Gram-negative bacteria.
Gram-positive bacterium used in the present study was B. subtilis.
While, Gram-negative strain investigated was E. coli. All the bacte-
rial strains were procured from Institute of Microbial Technology,
Chandigarh, India.
3
60–550 nm after 24 h of equilibration. The excitation and emis-
sion slit widths of 2.5 and 2.5–4.0 nm, respectively, were used for
the fluorescence measurements. The background spectra, either of
the buffer alone or of the buffer containing HMP were subtracted
from the corresponding sample spectra in all experiments.
A PerkinElmer LS-55 spectrophotometer equipped with filter
polarizers that use the L-format configuration was used for fluo-
rescence anisotropy measurements of DPH probe (details could be
found elsewhere) (Roy et al., 2005). An average of six measure-
ments was taken to calculate the final fluorescence anisotropy (r)
value. The solution temperature of the samples was controlled by
Thermo Neslab RTE-7 circulating water bath, which is connected
directly to the magnetically stirred cell holder in the spectrometer.
Culture conditions: Investigations of antibacterial activities were
performed by both broth dilution and spread plate method (Pitt
et al., 1994; Ikeda et al., 1984; Mitra et al., 2009; Roy and Das, 2008).
The LB medium was utilized as a liquid medium that contained
tryptone (10 g), yeast extract (5 g) and NaCl (10 g) in 1000 mL sterile
distilled water at pH 7.0. Besides, LB agar that contained tryptone
(10 g), yeast extract (5 g), NaCl (10 g) and agar (15 g) in 1000 mL of
sterile distilled water of at pH 7.0 was used as a solid medium for
spread plate study. The stock solutions of all the polymers as well
as the required dilutions were made in autoclaved sterile water.
For each bacterium, a representative single colony was picked up
with a wire loop and that loop-full of culture was spread on LB agar
◦
2
.8. Dynamic light scattering measurements
slant to give single colony and incubated at 37 C for 24 h. These
fresh overnight cultures of all the bacteria were diluted as required
6
9
The dynamic light scattering (DLS) experiments were carried out
to give a working concentration in the range of 10 -10 colony
forming units (cfu)/mL before every experiment.
on a home-built light scattering spectrophotometer (Dutta et al.,
009). The incident beam was generated from a vertically polar-
ized 100 mW He–Ne laser source (ꢁ = 532 nm) fixed at one arm of a
2
Determination of minimum inhibitory concentration (MIC): The
MIC is defined as the lowest concentration of an antimicrobial agent
that will inhibit the growth of a microorganism after incubation.
MIC values of HMPs were estimated by both broth dilution and
spread plate method. MIC was measured using a series of test tubes
containing the HMPs (0.05–200 ␮g/mL) in 5 mL LB broth. Diluted
bacterial culture was added to each test tube in identical con-
◦
◦
◦
◦
goniometer, at varying scattering angles (ꢂ = 50 , 70 , 90 , 100 , and
◦
1
(
20 ). The scattered beam was counted by a photo-multiplier tube
PMT) detector, mounted on other arm of the goniometer. The out-
put current from the PMT was then suitably amplified and digitized
through various electronics before it was fed to a 256-channel dig-
ital correlator (7132 Malvern, UK) with 50 ns initial delay time. The
7
8
centration, which was 7.5 × 10 − 1 × 10 cfu/mL for B. subtilis and
◦
7
7
temperature was set at 25 C, unless changed to set other values.
3.75 × 10 − 7.5 × 10 cfu/mL for E. coli. All the test tubes were then
◦
Prior to the measurement, each solution was cleaned by filtration
through a 0.45 ␮m filter paper (Millipore Millex syringe filter). The
final solution was loaded into an optical quality cylindrical quartz
sample cell and placed in a borosilicate glass cuvette containing an
incubated at 37 C for 24 h. The optical density of all the solutions
was measured before and after incubation at 650 nm. The LB broth
containing bacteria was used as a positive control. For compari-
son, both Gram-positive and Gram-negative bacteria were tested
in broth supplemented with classical antibiotic streptomycin. All
◦
index matching liquid (trans-decalene) at 25 C for 30 min. The cor-

3
02
P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
the experiments were performed in triplicate and were repeated
twice. The antimicrobial activity was also tested through spread
plate method. For these experiments, an equal amount of bacterial
culture was added to test tubes containing desired concentration
of polymer solution in liquid LB medium. The test tubes were then
◦
inoculated at 37 C for 5–7 h. Then, 50 ␮L from each tube was spread
onto LB agar plates inside laminar flow. Finally, plates were incu-
◦
bated at 37 C for 24 h, and the viable cells were counted.
2.12. Cytotoxicity assay
Cell culture: The 3T3 cell line (mouse fibroblasts) was grown
in RPMI-1640 medium supplemented with 10% FBS, 2 mM l-
glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin.
◦
Cells were grown in T-25 flasks at 37 C, 5% CO2 with a feeding
cycle of 2 days. After cells became 80% confluent (after 2 days) they
were trypsinized (0.25% Trypsin + 0.1% EDTA), centrifuged, and sus-
pended in RPMI. Cells were washed twice and finally were frozen
under liquid nitrogen in RPMI containing 10% dimethyl sulfoxide
Fig. 2. (A) 1H NMR spectra of typical poly(DMAEMA-DA) copolymer in CDCl3 (with
0 ␮L CD3OD in 0.5 mL); (B) GPC chromatograms of (a) poly(DMAEMA-HA), (b)
poly(DMAEMA-OA), (c) poly(DMAEMA-DA).
(
DMSO) for future use. For subsequent passages, cells were seeded
3
3
2
in fresh T-25 flasks at a density of 3 × 10 cells/cm and were cul-
tured in RPMI with a feeding cycle of 2 days.
MTT assay: The HMPs were dissolved in sterile water (pH 7.4)
and filtered through 0.2 ␮m polycarbonate filter. Then the sam-
ples were diluted to desired concentrations. Cell suspensions were
Hemolytic effect of each treatment was expressed as percent cell
lysis relative to the untreated control cells (% control) defined as:
3
seeded into a 96-well plate at 3 × 10 cells/well in 0.1 mL complete
hemolysis (%)
medium. The cells were allowed to adhere and grow for 24 h at
−
(
Abs at 550 samples − Abs at 550 Ve control)
◦
3
7 C in an incubator (Heraeus Hera Cell), after which the medium
= (
× 100
(2)
+
−
Abs at 550 Ve control − Abs at 550 Ve control)
was aspirated and replaced with 0.1 mL fresh medium containing
control and samples with the desired concentrations. After 48 h, the
culture medium was removed, the cells were washed with sterile
phosphate buffer saline (PBS) (0.1 mL/well) three times. Cell viabil-
ity was assessed using a conventional MTT dye reduction assay. A
2.14. Statistical analysis
The statistical method used to analyze the significant differ-
0
4
.1 mL of MTT reagent in PBS (1 g/L) was added to each well. After
–5 h incubation, the MTT reagent mixture was gently removed.
ences between control and treatment groups is one-way analysis of
variance (ANOVA). The results were expressed as mean ± SD unless
otherwise noted; p < 0.001 was considered statistically significant.
Then 0.1 mL DMSO was added into each well to dissolve the pur-
ple formazan precipitate which was reduced from MTT by the
viable cells with active mitochondria. The formazan dye was mea-
sured spectrophotometrically using benchmark microplate reader
at 550 nm. All assays were performed in triplicate. The cytotoxic
effect of each treatment was expressed as percent cell viability
relative to the untreated control cells (% control) defined as:
3. Results and discussion
3.1. Molecular characterization of the copolymers
To demonstrate the successful syntheses of the random
cell viability (%)
1
copolymers FT-IR, and H NMR spectra were studied. Disap-
−
1
(
absorbance at 550 of treated cells with samples)
pearance of the C C bond stretching frequency (1610 cm
)
= (
absorbance at 550 of control cells without samples)
×100 (1)
of the acrylate monomers in the FT-IR spectrum (not shown
here) indicates the formation of polymeric structures. This was
further confirmed by the absence of the vinylic proton signal
1
2.13. Hemolytic assay
along with broad peaks in the H NMR spectrum. A represen-
tative 1H NMR spectrum of poly(DMAEMA-DA) is presented in
Fig. 2(A). The signals at 4.3 and 2.8 ppm correspond, respec-
tively, to the proton bonded to the carbon adjacent to oxygen (br,
2H, –O–CH –CH –NH(CH ) ) and to the methyl groups attached
The copolymers were dissolved in sterile water to desired con-
centrations (0.1 g/L, 1 g/L). Hemocompatibility was analyzed using
the protocol reported by Katanasaka et al. (2008) with some modi-
fication. In brief, blood was obtained from 6-week-old BALB/c male
2
2
3 2
to nitrogen (br, 6H, –O–CH –CH –NH(CH ) ) of the DMAEMA
2
2
3 2
mice and red blood cells (RBC) were collected by centrifugation
monomer. The peaks of the DA monomer are observed at
1.6 ppm (br, 18H, –O–CH –CH –(CH ) –CH ), and 0.8 ppm (m,
◦
(
1500 rpm, 5 min, and 4 C) of the blood. The collected RBC pellet
2
2
2
9
3
was diluted in 20 mM HEPES buffered saline (pH 7.4) to give a 5%
v/v) solution. The RBC suspension was added to HEPES-buffered
3H, –O–CH –CH –(CH ) –CH ). The signals corresponding to the
2 2 2 9 3
(
methylene and methyne protons on the main chain are observed
from 1.8 to 2.5 ppm. The integrated signals at 4.3 ppm for DMAEMA,
and either of 1.6 ppm or 0.8 ppm for DA were employed to cal-
culate the mole fraction of the two monomers in the copolymer.
The results agree very well with the feed composition, taking the
experimental error (± 5%) of the method into account.
saline, 1% Triton X-100, and samples and incubated for 60 min at
◦
3
7 C. After centrifugation with Heraeus table top centrifuge 5805R
◦
at 12,000 rpm at 4 C, the supernatants were transferred to a 96-
well plate. Hemolytic activity was determined by measuring the
absorption at 550 nm using benchmark microplate reader (Biorad
Microplate reader 5804R). The samples with 0% lysis (HEPES buffer
The GPC experiment was performed to measure the molar
masses of the copolymers. Representative overlaid GPC chro-
matogram traces of poly(DMAEMA-HA), poly(DMAEMA-OA), and
+
saline) and 100% lysis (1% Triton X-100) were referred as Ve and
−
Ve control, respectively. All assays were performed in triplicate.

P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
303
Table 1
Fig. S2 of “supplementary material” were linearized applying Fuöss
equation:
Copolymer composition, molecular weights, and polydispersity index (PDI), intrinsic
viscosity ([ꢃ]), and limiting surface tension (ꢀmin) of the copolymers.
ꢃsp
[ꢃ]
1 + kC0.5
Copolymer
Mw/104
g/mol)
Mn/104
(g/mol)
PDI
[ꢃ] (L/g)
ꢀmin
=
(3)
C
(
(mN/m)
Poly(DMAEMA-HA)
Poly(DMAEMA-OA)
Poly(DMAEMA-DA)
6.38
6.75
7.19
4.34
4.19
4.70
1.47
1.61
1.53
0.102
0.162
0.202
55.1
52.7
50.3
where ꢃsp, C [ꢃ] and k are the specific viscosity, the concentration
of the polymer, the intrinsic viscosity, and Fuöss constant, respec-
tively. Viscometric data obtained in terms of the Fuöss equation are
presented in the inset of Fig. S2 of “supplementary material”. As can
be observed straight lines are obtained for both the samples over
a wide range of concentration allowing us to calculate the intrin-
sic viscosity values. The intrinsic viscosity values determined using
Fuöss method is presented in Table 1. It is observed that [ꢃ] value is
higher for the polymer with longer hydrophobic chain length. This
is quite reasonable since the viscosity reflects the polymer size,
larger the size of the polymer, higher is the viscosity.
poly(DMAEMA-DA) have been shown in Fig. 2(B). All three copoly-
mers exhibit unimodal molecular weight distribution. The average
molecular weight and polydispersity index (PDI) obtained are sum-
marized in Table 1. The data in Table 1 suggest that the HMP with
longer hydrophobic chain has higher molecular weight compared
to the copolymer of shorter hydrophobic chain analog.
Aqueous solutions of the HMPs became turbid beyond pH 9.
Increased hydrophobicity of these copolymers is likely responsible
for the precipitation from aqueous solution at higher pH. The HMPs
are hydrochloride salts of the corresponding polymer containing
tertiary amine group along its backbone. As mentioned earlier the
average pKa value of the ammonium groups of poly(DMAEMA) as
reported in the literature is ∼7.9 (Merle, 1987; Pradny and Sevcik,
3.3. pH dependent self-aggregation of the HMPs
The copolymers are carrying tertiary amine groups along their
backbone. It is therefore obvious that the conformation of the HMPs
poly(DMAEMA-HA), poly(DMAEMA-OA), and poly(DMAEMA-DA)
should be a function of the solution pH. The pH dependent con-
formational transition was monitored by measuring the spectrum
of NPN fluorescence in the presence of copolymers at different
solution pH. The fluorescence emission spectrum of NPN is known
to depend on the environment of the system. Generally, a very
weak fluorescence is detected in aqueous medium with emission
maximum (ꢁmax) at 460 nm which gets blue shifted accompa-
nied by a huge increase of fluorescence intensity in going from
water to less polar solvent. The detailed spectral properties of
this hydrophobic probe have been reported in our earlier publi-
cations (Dutta et al., 2009; Mohanty and Dey, 2007). The shift of
emission maximum (ꢄꢁ = ꢁmax(water) − ꢁmax(polymer)) and the
change in relative intensity (I/Io) are shown in Fig. 3. As seen, both
ꢄꢁ (Fig. 3(a)) and I/Io ((Fig. 3(b)) increases in going from lower to
higher pH. The feature of the curves is nearly sigmoid. Although a
large change in ꢄꢁ was observed for copolymers poly(DMAEMA-
HA) and poly(DMAEMA-OA), much less change can be found for the
copolymer poly(DMAEMA-DA). At low pH (<7.0), the amine groups
become protonated thus increasing the overall charge along the
backbone. As a result, the hydrophobic interaction among the long
chain alkyl groups is weakened by the charge repulsion causing
release of the probe to bulk water. In other words, the aggregate
structures are disrupted as a result of lowering the pH. However,
at higher pH (>7.0) the free amine groups become less protonated
and the hydrophobic character along the chain becomes stronger.
Consequently, a large fraction of the probe molecules further parti-
tioned into the hydrophobic microenvironments. It is quite evident
from the plots of both figures that all the three HMPs have a phase
transition around pH 6.0 and may be taken as the average pKa
value of the tertiary amine groups. Therefore, the zeta-potential
(ꢅ) measurement was performed for the copolymers at pH 5.0 for
1 g/L copolymer solution and the data are summarized in Table 2.
The positive potential values clearly suggest that the polymers are
positively charged.
1
985). This means that in neutral pH the HMPs exist in the par-
tially protonated form, whereas in pH < 7.0 the amine groups are
expected to be completely ionized. The change in pH can there-
fore change the conformation of the HMPs in solution and has
been discussed below. In their partially charged form at neutral
pH the HMPs are expected to remain in the conformation that
favors intra-polymer aggregation producing unimolecular micelles.
On the other hand, in their fully ionized form in pH < 7.0, they
are expected to behave either like polyelectrolytes or to undergo
inter-polymer aggregation. Such conformational changes of the
copolymers can be used in pH-triggered intracellular delivery of
drugs and DNA by exploitation of intracellular pH gradients which
varies between 4.5 and 8 among different organelles (cytoplasm,
endosomes, lysosomes, endoplasmic reticulum, mitochondria, and
nuclei) (Asokan and Cho, 2002). For this reason, we have stud-
ied their physicochemical properties in pH 5 to ensure complete
ionization of the amine groups.
3
.2. Surface activity and solution viscosity of the HMPs
Because of the amphiphilic character, the HMPs are expected to
possess surface-active properties. This was evaluated by monitor-
ing the equilibrium surface tension (ꢀ) of water at different polymer
concentrations. As observed in Fig. S1 of “supplementary mate-
rial”, the surface tension of water, ꢀ decreases with increase in
concentration indicating their amphiphilic character. The ꢀmin val-
ues of the HMPs are included in Table 1. The data suggest that the
amphiphilicty increases with the increase of chain length of the
hydrophobe unit.
The variation of reduced viscosity (ꢃsp/C) with the polymer con-
centration in aqueous buffer solution (pH 5) has been shown in
Fig. S2 of “supplementary material”. All the copolymers exhibit a
typical polyelectrolyte behavior in dilute solution. That is the ꢃsp/C
value increased with decreasing (copolymer) due to the expan-
sion of the macroion chain, which is caused by the intra- and
inter-molecular repulsive interactions among the ionized groups
along the chain. Reduced viscosity values, at the same polymer
concentration are seen to increase in the order: poly(DMAEMA-
HA) < poly(DMAEMA-OA) < poly(DMAEMA-DA), which means for a
constant degree of polymerization, with increasing the hydropho-
bic chain length a higher content of the cationic units in the
copolymer is responsible for an increase of the coil dimen-
sion. To obtain intrinsic viscosity [ꢃ] value the curves shown in
3.4. Self-assembly behavior and microenvironments of the
aggregates
To obtain information about the aggregation properties of the
HMPs, steady state fluorescence probe studies were carried out
next in aqueous solution of pH 5 using NPN, pyrene, and DPH
as probe molecules. The fluorescence spectra of NPN were fur-
ther measured in the presence of different polymer concentration.
The emission maximum of NPN undergoes a blue shift accom-

3
04
P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
◦
Fig. 3. Plots of (a) ꢄꢁ, and (b) I/Io of NPN probe as a function of pH in the presence of 0.25 g/L copolymer solution at 30 C: (ꢀ) poly(DMAEMA-HA), (᭹) poly(DMAEMA-OA),
and (ꢁ) poly(DMAEMA-DA).
Table 2
Physico-chemical properties of the copolymers (1 g/L) in aqueous acetate buffer solution (pH 5).
CACa (g/L)
ꢄꢁb (nm)
I1/I3b
rc
ꢁRhꢂ (nm)
d
ꢅe (mV)
Copolymer
Poly(DMAEMA-HA)
Poly(DMAEMA-OA)
Poly(DMAEMA-DA)
0.0455
0.0366
0.0169
17
24
31
1.69
1.6
1.42
0.178
0.184
0.197
16, 223
14.5, 158
8, 140
+32.2
+28.3
+24.5
a
Critical aggregation concentration (CAC).
Micropolarity (ꢄꢁ and I1/I3).
Viscosity (r).
b
c
d
e
Mean hydrodynamic diameter ꢁRhꢂ.
Zeta potential (ꢅ).
panied by a rise in fluorescence intensity in the presence of
increasing concentration of copolymer, indicating partitioning of
NPN into the hydrphobic domains. The shifts of emission max-
imum (ꢄꢁ) determined at different polymer concentrations are
plotted in Fig. 4(a). Both the relative fluorescence intensity (I/Io)
ing the micropolarity of the aggregates using pyrene. The I /I ratio
1 3
−
4
determined in the concentration range 1 × 10 to 4 g/L for all the
HMPs are presented in the inset of Fig. 4(a). It is observed that above
a certain concentration, the I /I3 ratio decreases sharply, indicat-
1
ing intermolecular interaction. The concentration corresponding to
the inflection point of the sigmoidal curves in the inset of Fig. 4(a)
are close to the value obtained from the fluorescence titration of
(
not shown), and ꢄꢁ was found to increase with increase in
polymer concentration. As can be seen in Fig. 4(a), a concentration-
independent regime of ꢄꢁ value appeared in all cases. However,
with the increase in polymer concentration the value of ꢄꢁ grad-
ually increases. The concentration corresponding to the inflection
point of the plots as indicated in Fig. 4(a) was therefore taken as
CAC. The CAC value thus obtained are presented in Table 2. The
CAC values are in the order poly(DMAEMA-HA) > poly(DMAEMA-
OA) > poly(DMAEMA-DA). This is just opposite to the order of
molecular weights of the copolymers. The ꢄꢁ value, which is a
measure of the hydrophobicity of the aggregates was determined
at a given concentration (1.0 g/L) of the all the copolymer solutions.
The data listed in Table 2 show that hydrophobicity increases with
the increase of molecular weight. The formation of hydrophobic
domains at very low concentration was also confirmed by measur-
NPN and can therefore be regarded as CAC. The I /I3 value (∼1.35)
1
obtained for poly(DMAEMA-DA) at the limiting concentration is
very similar to those observed for CTAC or CTAB surfactant micelles
(Kalyanasundaram, 1987). The I /I3 value in 1 g/L polymer solution
1
is given in Table 2. The I /I3 value of the HMPs decreases with the
1
increase of hydrophobe chain length, which is consistent with the
increase of ꢄꢁ value of the NPN probe.
To further investigate the microenvironment of the copolymer
aggregates and its relation with the hydrophobic chain length,
fluorescence intensity as well as fluorescence anisotropy (r) mea-
surements were performed employing DPH probe. The DPH is
almost insoluble in water and thus weakly fluorescent. However,
its fluorescence intensity is greatly enhanced upon solubilization in
◦
Fig. 4. (a) Plots of emission maximum shift (ꢄꢁ = ꢁmax(water) − ꢁmax(polymer)), of NPN versus polymer concentration at 30 C. (Inset: Plot of intensity ratio (I1/I3) of pyrene
◦
against polymer concentration]; (b) Plots of relative fluorescence intensity (I/Io) of DPH probe in different polymer concentration at 30 C (Inset: Variation of fluorescence
anisotropy (r) of DPH probe with the concentration; (ꢀ) poly(DMAEMA-HA), (᭹) poly(DMAEMA-OA), and (ꢁ) poly(DMAEMA-DA).

P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
305
Fig. 5. TEM images (unstained) of 1 g/L (A) poly(DMAEMA-HA) and (B) poly(DMAEMA-DA) in acetate buffer of pH 5.
the hydrophobic microdomains of micelles. Therefore, the intensi-
ties of DPH fluorescence were measured at an emission wavelength
of 450 nm in aqueous copolymer solution and were compared with
the intensity of DPH in water. In poly(DMAEMA-DA), the relative
intensity (I/Io) increases considerably with the increase of poly-
mer concentration (Fig. 4(b)). However, the increase is much less
at the same polymer concentration for poly(DMAEMA-HA), and
poly(DMAEMA-OA). These observations also signify the partition-
ing of DPH into the hydrophobic microdomains formed through
association of pendent alkyl chains of the copolymers. Here also,
a concentration-independent region was observed which further
substantiate the intermolecular association of the copolymers. The
rotational motion of DPH molecule usually becomes restricted
when it is solubilized into the hydrophobic core of the aggregate.
This is evident by the high r-values of DPH in solutions of the
HMP at concentrations above the respective CAC value. The plots
of r as a function of (copolymer) have been shown in the inset
of Fig. 4(b). As observed, the r-value is independent of polymer
concentration above CAC. The r-values in 1 g/L polymer solution
are listed in Table 2. It is important to note that the r-values
for all the three polymers at any polymer concentration above
CAC is very high and increases with the increase of hydrophobe
chain length, suggesting tighter packing of the hydrophobes in
the aggregate with the increase of chain length. This restricts
the motion of the DPH molecule resulting in a high value of r.
The constant value of r at higher polymer concentrations might
be due to the aggregation of smaller aggregates forming larger
aggregates.
3.5. Microstructure of the aggregates
The morphology of the aggregates formed by the HMPs in
aqueous solution was visualized by TEM technique. The images
of the unstained samples (1 g/L) of poly(DMAEMA-HA), and
poly(DMAEMA-DA) have been presented in Fig. 5. In both cases, the
spheroid morphology of the copolymer aggregates is clearly visible.
The sizes of the micelles observed for poly(DMAEMA-HA) are larger
(∼100–150 nm) than those formed by poly(DMAEMA-DA). How-
ever, with poly(DMAEMA-DA) a large number of smaller aggregates
of different sizes are visible, and owing to the stronger interaction
among hydrophobes they seem to be merging together. The parti-
cles, as expected, are positively charged, which is confirmed by the
zetapotential (ꢅ) values (Table 2).
3.6. Hydrodynamic diameter of the aggregates
Dynamic light scattering experiments were performed to mea-
sure the effective hydrodynamic radii and the population (in terms
of size) of the aggregates formed by the HMPs. Fig. 6(a) illustrates
the histograms of size distribution of 2 g/L copolymer in pH 5
containing 100 mM NaCl. For all the three HMPs, a bimodal size
distribution was observed. As seen from Fig. 6, poly(DMAEMA-HA)
◦
Fig. 6. (a) Intensity average distribution for 2 g/L copolymer in aqueous solution of pH 5 (with 100 mM NaCl) at 25 C; (a) poly(DMAEMA-HA), (b) poly(DMAEMA-OA), (c)
poly(DMAEMA-DA). Average hydrodynamic radius (ꢁRhꢂ) versus copolymer concentration (a) ꢁRhꢂfast, (b) ꢁRhꢂslow; (ꢀ) poly(DMAEMA-HA), (᭹) poly(DMAEMA-OA), and (ꢁ)
poly(DMAEMA-DA).

3
06
P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
Fig. 7. (a) Plots of I1/I3 of pyrene and (b) emission maximum shift (ꢄꢁ) of NPN at different NaCl concentrations for 0.1 mg/mL copolymers; (ꢀ) poly(DMAEMA-HA), (᭹)
poly(DMAEMA-OA), and (ꢁ) poly(DMAEMA-DA).
has comparatively narrow and monodispersed size distribution
with the population of a mean hydrodynamic diameter about
concentrations, showing a preference for effective interaction of
hydrophobic groups. Thus, for poly(DMAEMA-DA) copolymer, the
4
40 nm, which can be attributed to intermolecular association
ꢁR ꢂ
reduces to a value of ∼8 nm from 13 nm. The hydrodynamic
h
fast
forming larger aggregates and the remaining population exhib-
ited smaller size of about 34 nm, ascribed to an aggregate of small
number of polymer chains. However, with the increase in chain
length of the hydrophobe in the copolymer the mean diameter
for both the population gradually decreases and the distribution
becomes broad, indicating stronger hydrophobic association. Thus
for poly(DMAEMA-DA), the average diameter reduces to ∼300 and
radius corresponding to the slow decay mode ꢁR ꢂ
, on the other
h
slow
hand, exhibits a different behavior. In dilute solution, a gradual
decrease in ꢁR ꢂ value followed by an increase at higher concen-
h
trations can be observed. Since in very dilute solution the HMPs
are mostly hydrated, it is quite obvious to exhibit a much larger
hydrodynamic radius. However, as soon as the polymer concen-
tration increases, the inter-chain hydrophobic interaction favors
micelle-like aggregate formation, which causes gradual decrease
in hydrodynamic radius (Fig. 6(b)). Further, increase in hydrody-
namic radius occurs probably due to the formation of multicore
multipolymer aggregates.
∼
20 nm for the larger and smaller aggregates, respectively.
The relaxation rates (ꢆ ) were also measured at various scat-
◦
tering angles in the range of 50–120 for the same concentration
for all three HMPs. For both the fast and slow relaxation modes,
ꢆ is plotted as a function of the scattering wave vector and are
presented in Fig. S3 (“supplementary material”). The linear rela-
3.7. Effect of salt concentration
2
tionship of ꢆ with q can be found for both modes. The straight lines
pass through the origin, suggesting that the relaxation modes are
virtually due to the translational diffusion of the scattering particles
Addition of salt causes changes in conformation of the poly-
electrolyte. Usually, the electrolyte screens the ionic counterpart
of the hydrophilic group and in turn facilitates the chain entangle-
ment/packing. As a result, the aggregation through hydrophobic
group becomes more favored. To investigate the effect of elec-
trolyte for these copolymer conformations, we have performed
the fluorometric titration using NPN and pyrene against sodium
chloride concentration (a monovalent electrolyte) in 0.1 g/L copoly-
that are spherical in shape. Average hydrodynamic radius (ꢁR ꢂ) was
h
calculated from the Stokes–Einstein relation using values of diffu-
2
sion coefficient (D) estimated from the slope of the ꢆ − q plots
(
for both the fast and slow modes). Values of ꢁR ꢂ thus obtained
h
for 1 g/L copolymer solution are listed in Table 2. The DLS mea-
surements were also performed at different concentrations of the
HMPs. The ꢁR ꢂ determined at various concentrations are presented
mer. The plots of I /I3 and ꢄꢁ at different NaCl concentrations
h
1
in Fig. 6(b). As seen, the radius corresponding to the fast decay mode
are shown in Fig. 7. Indeed, a gradual decrease of I /I3 and
1
(
ꢁR ꢂ ) for poly(DMAEMA-HA) has a value of ∼17 nm in dilute
increase of ꢄꢁ value clearly demonstrate the profound associ-
h
fast
solution, which remained unaltered within the studied concentra-
tion range (Fig. 6(b)). However, with the increase in hydrophobe
chain length, a decreasing trend of ꢁRhꢂfast was observed at higher
ation of the hydrophobic group caused by the shielding of the
−
charges by the negatively charged Cl ions. The stronger asso-
ciation at high salt concentrations was further supported by the
◦
Fig. 8. Size distributions at different NaCl concentrations for 0.1 g/L (a) poly(DMAEMA-HA), (b) poly(DMAEMA-OA), and (c) poly(DMAEMA-DA) copolymer at 25 C.

P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
307
Fig. 9. Antibacterial assessment images of copolymers against B. subtilis; (A) poly(DMAEMA-HA), (B) poly(DMAEMA-OA), (C) poly(DMAEMA-DA).
measurement of DLS at the same copolymer concentration with
three different NaCl concentrations. Intensity average size distri-
butions of the aggregates in copolymer solutions at different NaCl
ever, with increasing temperature they disintegrate into individual
polymer chains, as reflected by the lower values of r at higher
◦
temperatures. The melting temperature is greater than 37 C
(
0.3, 0.6, 1.0 M) concentrations have been depicted in Fig. 8. It is
and increases in the order poly(DMAEMA-DA) > poly(DMAEMA-
OA) > poly(DMAEMA-HA).
observed that at low NaCl concentration (0.3 M), the copolymer
poly(DMAEMA-HA) exhibits a monomodal distribution, whereas
the HMPs poly(DMAEMA-OA) and poly(DMAEMA-DA) show a
bimodal distribution with relatively larger diameters. However,
with increasing NaCl concentration the distribution shifted to lower
diameter range accompanied by a broadening the peak width,
which confirms stronger hydrophobic association.
3.9. Antibacterial studies
The antibacterial activities of the copolymers poly(DMAEMA-
HA), poly(DMAEMA-OA), and poly(DMAMEA-DA) were investi-
gated against both the Gram-positive (B. subtilis) and Gram-
negative bacteria (E. coli). The pictures of B. subtilis and E. coli before
(control) and after treatment with the HMP (above and below MIC)
are shown in Figs. 9 and 10. The minimum inhibitory concentra-
tions (MIC) determined for each HMP against B. subtilis and E coli
bacteria are summarized in Table 3. As seen, all the three HMPs
under investigation have biocidal activity to Gram-positive bacte-
ria B. subtilis, but in the case of Gram-negative bacteria E. coli, only
poly(DMAEMA-HA), and poly(DMAEMA-OA) were active with MIC
values 50 and 200 ␮g/mL, respectively. In contrast, poly(DMAEMA-
DA) remained ineffective up to a concentration of 200 ␮g/mL. It
has long been known that the chain length of hydrophobic unit
3
.8. Effect of temperature
Thermal response of the copolymer micelles was investigated
by fluorescence probe technique using DPH probe. Fluorescence
anisotropy (r) of DPH was measured to investigate the effect of
temperature on the micellar aggregates of the HMPs. Fig. S4 of “sup-
plementary material” shows the variation of r as a function of
temperature in 0.25 g/L copolymer solution. As observed, below
3
◦
0 C, the HMPs have high r-values, which suggest that the
micelles are very rigid and stable at lower temperature. How-
Fig. 10. Antibacterial assessment images of copolymers against E.coli; (A) poly(DMAEMA-HA), (B) poly(DMAEMA-OA).

3
08
P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
Chart 1. Structure of polymer-ct-DNA complex in solution.
also be the case with poly(DMAEMA-DA) copolymer as it contains
Fig. 11. Stern–Volmer plots for fluorescence quenching by copolymers. (ꢀ)
poly(DMAEMA-HA), (᭹) poly(DMAEMA-OA), and (ꢁ) poly(DMAEMA-DA).
longer alkyl chain (C12) compared to poly(DMAEMA-HA) (C ) and
6
poly(DMAEMA-OA) (C ) copolymers. The excessive hydrophobicity
8
resulted from poly(DMAEMA-DA) might possibly cause aggrega-
tion, reducing the number of cationic charges available to interact
and thus making it difficult to kill the E. coli.
in the cationic polymer plays a profound impact on the antibac-
terial activities (Ignatova et al., 2004; Kanazawa et al., 1993). To
gain an effective antimicrobial action, an optimal range of alkyl
chain is required (usually in the ranges of C6 to C18) as it can
strongly bind to the cytoplasmic membrane of the bacteria which
constitutes long-chain fatty acid with 12–20 carbon units through
hydrophobic interaction (Domagk, 1935; Goodson et al., 1999;
Franklin and Snow, 1981). Palermo and Kuroda reported recently
the antibacterial activity of a library of amphiphilic random copoly-
mers against E. coli containing DMAEMA monomers with various
Further from the results of MIC values (Table 3), it can be
concluded that these HMPs have a greater killing efficiency for
Gram-positive bacteria than Gram-negative bacteria. This is in
accordance with the result reported in the literature (Ignatova
et al., 2004; Lenoir et al., 2006). Since, the Gram-positive bac-
teria have only peptidoglycan cell wall, the penetration for the
cationic polyelectrolytes with hydrophobic group are easier. How-
ever, in the case of Gram-negative bacteria (E. coli), the cells are
being surrounded by an additional outer membrane and hence,
it is more difficult for macromolecules to diffuse through the cell
wall. Furthermore, it is worth noting that among the three HMPs,
poly(DMAEMA-OA) with octyl chain (C8) as hydrophobic group
has the strongest microbial efficiency with MIC value 20 ␮g/mL for
Gram-positive B. subtilis which is just opposite to E. coli. There-
fore, the poly(DMAEMA-OA) copolymer plausibly maintained the
suitable balance of cationic charge with hydrophobicity for effec-
tive antimicrobial action against Gram-positive bacteria. In fact,
like ours many other groups have also established that cationic
polymers with octyl chain do possess optimal balance between
the hydrophobicity and bacterial efficacy leading to highest activity
(Ignatova et al., 2004; Lenoir et al., 2006; Roy et al., 2008).
ratios of methylmethacrylate (C ) (from 0 to 70 mol%) or butyl-
1
methacrylate (C ) (from 0 to 40 mol%) (Palermo and Kuroda, 2009).
4
They observed that the copolymers consisting of methyl (C ) side
1
chain as hydrophobic group with less than 20 mol% content failed
to completely inhibit the growth of E. coli even up to 2000 ␮g/mL
and reached a minimum of 242 ␮g/mL in the MIC value at 50%
hydrophobe concentration and then increased as the hydrophobe
concentration increased. In contrast, the copolymer with butyl (C4)
side chain as hydrophobic group were very effective in killing E. coli
even at 12 mol% with MIC value 24 ␮g/mL. Similarly, in the case of
polymethacrylamide random copolymer bearing protonated pri-
mary amine groups, the hexyl side chain containing copolymers
impart more potent antimicrobial activity as compared to butyl
side chain containing polymers at similar degree of polymeriza-
tion and identical hydrophobe concentration (Palermo et al., 2009).
To establish a relationship between the effect of hydrophobicity of
the alkyl chain and their antimicrobial activities, Roy et al. (2008)
further investigated a series of polymer on cellulosic filter using
DMAEMA monomer which was subsequently quaternized with
alkyl bromides of different chain lengths (C –C16). They observed
8
that cellulose graft copolymer that has been quaternized with a
shorter alkyl chain (C ) bromide is more effective against E. coli
8
as compared to the cellulose graft copolymers quaternized with
longer alkyl chain (C12 and C16) bromides. Therefore, this may
Table 3
Minimum inhibition concentration of cationic polymers (␮g/mL) and Stern–Volmer
constant values for binding with ct-DNA of cationic polymers.
Copolymer
MIC (␮g/mL)
KSVa (L/g)
kqb
(
×10− L/g/s1)
9
B. subtilis
E. coli
poly(DMAEMA-HA)
poly(DMAEMA-OA)
poly(DMAEMA-DA)
50
20
35
50
200
>200 ␮g/mL
40
62.7
88.7
1.81
2.85
4.02
Fig. 12. MTT assay based fibroblast cell line 3T3 cell viability (in %) as a function
of concentration for cationic copolymers at physiological pH (7.4). Significant dif-
ference is shown as ***p < 0.001 versus control. The bars indicate the means ± SD.
(n = 3).
a
Stern–Volmer quenching constant.
Bimolecular quenching constant.
b

P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
309
Fig. 13. (A) The picture of hemolytic assay for the copolymers, (B) % hemolysis of copolymers at 0.1 g/L and 1 g/L concentration at physiological pH (7.4). Significant difference
−
is shown as ***p < 0.001 versus Ve control. The bars indicate the means ± SD. (n = 3).
3
.10. Complexation with calf-thymus DNA
In light of the above observations, the only way the increase of
hydrophobe chain length can increase the binding ability of the
HMP is through bilayer formation in solution. The ct-DNA becomes
sandwiched (Chart 1) between two bilayers formed by the HMP.
The appearance of turbidity in solution in the presence of larger
amount of ct-DNA is an indication of bilayer formation. It is well
known that the ability to form bilayer by amphiphilic molecules
is increased with the increase of hydrophobic chain length. This
means an increase of K value with the increase of the length of
hydrophobe unit of the HMP. Such a complexation mode has been
proposed by many authors for binding of phospholipids with DNA
(Bhattacharya and Bajaj, 2009).
In an attempt to examine the complextation ability of the
copolymers with DNA, fluorescence quenching studies were per-
formed for the copolymers with calf-thymus DNA (ct-DNA) using
EB as a fluorescent probe. The fluorescence spectra of EB (2 ␮M)
and EB-ct-DNA ([ct-DNA] = 0.01 g/L) in absence and in the pres-
ence of different concentrations of poly(DMAEMA-DA) have been
shown in Fig. S5 of “supplementary material”. As observed, EB has a
very weak emission band with an emission maximum of 590 nm in
water, but in the presence of ct-DNA its emission intensity is greatly
enhanced due to intercalation into the DNA strands. However,
adding increasing concentration of copolymer poly(DMAEMA-DA)
to EB-ct-DNA complex, the emission intensity of EB is significantly
quenched, which suggests binding of poly(DMAEMA-DA) with ct-
DNA.
3.11. Cytotoxicity assay
The biocompatibility of the three amine-based HMPs was eval-
uated in terms of MTT assays. The cytotoxicity of the copolymers
in the concentration range 0.05–2.0 g/L at physiological pH (7.4)
was evaluated by measuring the viability of 3T3 cells using the
MTT assay. As seen in Fig. 12, no significant toxicity was observed
at low concentration for all three HMPs. However, with increase
in concentration (>0.1 g/L) the cell viability significantly decreased
and a much higher toxicity was observed at concentration above
1.2 g/L. Therefore, the HMPs at higher concentrations are toxic
to this fibroblasts 3T3 cell line, while at lower concentration (i.e.
the concentration well above MIC) they did not affect the cell
growth and can be used for pharmaceutical applications. Unlike
hemolytic activity, the cytotoxicity of cationic polymers depends
differently. The greater number of cationic groups is considered
to be more cytotoxic and the cytotoxicity can be reduced to some
extent by increasing the hydrophobicity of cationic copolymer with
either increasing the hydrophobic chain length or increasing the
hydrophobe concentration. Palermo et al. recently investigated the
cytotoxicity of methacrylamide based random copolymers bear-
ing protonated primary amine and butyl or hexyl groups in the
side chains of different substitution against human epithelial Hep-2
cells (Palermo et al., 2009). They observed that in contrast to unsub-
stituted polymer, the butyl or hexyl substituted polymer showed
less cytoxicity up to 40 mol% hydrophobic substitutions which is
quite consistent to our findings.
The strength of binding of the HMP can be estimated by mea-
suring the equilibrium constant of binding process:
EB-ct-DNA + HMP = HMP-ct-DNA + EB
(4)
The extent of fluorescence quenching at various polymer con-
centrations can be used to determine the Stern–Volmer (S–V)
quenching constant (K), which is a measure of binding constant.
To determine the K value, the fluorescence intensity of EB-ct-DNA
was plotted against copolymer concentration (HMP) according to
S–V equation (Lakowicz, 2006):
Fo/F = 1 + K [HMP]
(5)
where Fo and F stand for the fluorescence intensities in the absence
and presence of polymer. The S–V plots for all the cationic HMPs
employed in this study have been presented in Fig. 11. The binding
constant K (Table 3) was obtained from the slope of the curve in
the linear range. The data suggest that the binding of copolymers
with longer hydrophobe chain is stronger than that with shorter
hydrophobe chain. This means that the hydrophobes in the polymer
backbone play a significant role in the binding process.
In order to understand the nature of binding of the cationic
copolymers with ct-DNA, the circular dichroism (CD) spectra of
DNA sample were measured in the presence of different concen-
trations of poly(DMAEMA-DA) as a representative HMP. The CD
spectra have been shown in Fig. S6 of “supplementary material”.
The CD spectrum of DNA showed very little change upon bind-
ing of the copolymer, suggesting that the duplex structure of DNA
remains almost unperturbed in the complexed state. This means
that the ct-DNA-HMP binding is purely electrostatic in nature.
3.12. Hemocompatibility studies
Compatibility of the copolymers has also been tested with
blood component red blood cells (RBCs). Hemolytic assays were

3
10
P. Dutta et al. / International Journal of Pharmaceutics 414 (2011) 298–311
performed for all the three copolymers poly(DMAEMA-HA),
poly(DMAEMA-OA), poly(DMAEMA-DA) in pH 7.4 (Hepes buffer,
2006/37/17/BRNS/235), and Ministry of Human Resource Develop-
ment (MHRD) of this work. PD thanks CSIR (09/081(0519)/2005-
EMR-I) for a research fellowship. The authors are thankful to
Dr. G. Ghosh, UGC-DAE Consortium for Scientific Research, BARC,
Trombay, Mumbai 400 085, India for assistance with the DLS mea-
surements.
2
0 mM). Freshly isolated BALB/c male mice RBC suspension (5%,
v/v) was added to HEPES-buffered saline, 1% Triton X-100 and
polymers with a final concentration of 0.1 and 1.0 mg/mL, and incu-
◦
bated for 60 min at 37 C. The pictures associated to the hemolytic
experiments are presented in Fig. 13(A). As seen although the
HMPs are compatible to 3T3 fibroblast cell line, these are incom-
patible to RBC at both lower and higher concentration. This is
also reflected by the % hemolysis presented in Fig. 13(B). Like
antibacterial activity and cytotoxicity, the hemolytic activity is also
strongly dependent on the chemical structures of polymers. Though
the increase in hydrophobic side chain is believed to cause an
increase in potency of antimicrobial effect of cationic polymers,
it is found that increasing the hydrophobicity eventually lead to
increased hemolysis. In this context, Palermo and Kuroda (2009)
have recently investigated in more detail the hemolytic activity of
amphiphilic random copolymers containing DMAEMA monomers
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ijpharm.2011.05.006.
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