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Cationic drug based self assembled polyelectrolyte complex micelles physicochemical, pharmacokinetic, and anticancer activity analysis

Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160

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Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb

Cationic drug-based self-assembled polyelectrolyte complex micelles:
Physicochemical, pharmacokinetic, and anticancer activity analysis
Thiruganesh Ramasamy a , Bijay Kumar Poudel a , Himabindu Ruttala a , Ju Yeon Choi a ,
Truong Duy Hieu a , Kandasamy Umadevi b , Yu Seok Youn c , Han-Gon Choi d ,
Chul Soon Yong a,∗ , Jong Oh Kim a,∗
a

College of Pharmacy, Yeungnam University, 214-1 Dae-dong, Gyeongsan 712-749, South Korea
St. Paul’s College of Pharmacy, Osmania University, Hyderabad, Telangana, India
c
School of Pharmacy, SungKyunKwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, 440-746, South Korea
d
College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, 55, Hanyangdaehak-ro, Sangnok-gu, Ansan 426-791,
South Korea

b

a r t i c l e

i n f o

Article history:
Received 22 March 2016
Received in revised form 31 May 2016
Accepted 2 June 2016
Available online 5 June 2016
Keywords:
Nanofabrication
Polyelectrolyte complex micelles
Cationic drugs
Pharmacokinetic
Anticancer activity

a b s t r a c t
Nanofabrication of polymeric micelles through self-assembly of an ionic block copolymer and oppositely
charged small molecules has recently emerged as a promising method of formulating delivery systems.
The present study therefore aimed to investigate the interaction of cationic drugs doxorubicin (DOX)
and mitoxantrone (MTX) with the anionic block polymer poly(ethylene oxide)-block-poly(acrylic acid)
(PEO-b-PAA) and to study the influence of these interactions on the pharmacokinetic stability and antitumor potential of the formulated micelles in clinically relevant animal models. To this end, individual
DOX and MTX-loaded polyelectrolyte complex micelles (PCM) were prepared, and their physicochemical
properties and pH-responsive release profiles were studied. MTX-PCM and DOX-PCM exhibited a different release profile under all pH conditions tested. MTX-PCM exhibited a monophasic release profile with
no initial burst, while DOX-PCM exhibited a biphasic release. DOX-PCM showed a higher cellular uptake
than that shown by MTX-PCM in A-549 cancer cells. Furthermore, DOX-PCM induced higher apoptosis
of cancer cells than that induced by MTX-PCM. Importantly, both MTX-PCM and DOX-PCM showed prolonged blood circulation. MTX-PCM improved the AUCall of MTX 4-fold compared to a 3-fold increase
by DOX-PCM for DOX. While a definite difference in blood circulation was observed between MTX-PCM
and DOX-PCM in the pharmacokinetic study, both MTX-PCM and DOX-PCM suppressed tumor growth
to the same level as the respective free drugs, indicating the potential of PEGylated polymeric micelles
as effective delivery systems. Taken together, our results show that the nature of interactions of cationic
drugs with the polyionic copolymer can have a tremendous influence on the biological performance of a
delivery system.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Conventional chemotherapeutic approach is the main treatment option for cancer [1]. Despite great strides made in
understanding cancer biology, conventional chemotherapeutic

drugs are characterized by non-specific distribution and high accumulation in healthy cells, leading to dose-limiting side effects
that seriously impede their clinical application [2]. To minimize
side effects and improve therapeutic efficacy of chemotherapeu-

∗ Corresponding authors.
E-mail addresses: csyong@ynu.ac.kr (C.S. Yong), jongohkim@yu.ac.kr (J.O. Kim).
http://dx.doi.org/10.1016/j.colsurfb.2016.06.004
0927-7765/© 2016 Elsevier B.V. All rights reserved.

tic drugs, various drug delivery systems have been developed.
Among them, block copolymer-based self-assembled polymeric
micelles have demonstrated promising potential in the delivery
of anticancer drugs. The nanosized micelles offer many advantages, including uniform size distribution, core-shell architecture,
high drug loading, and physical stability [3,4]. Polyethylene glycol
(PEG) is widely used to graft the hydrophobic part of amphiphilic
polymers and form the outer shell of the micelles. Such polymeric
micelles have been shown to increase the systemic circulation time
of drugs and preferentially accumulate in tumors via enhanced
permeability and retention (EPR) effect [5].
Polyelectrolyte complex micelles (PCM), a special class of
micelles formed by electrostatic interaction of opposite charged


T. Ramasamy et al. / Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160

species (ionic blocks), have recently been developed [6,7]. For drug
delivery applications, therapeutic moieties (small molecules, DNA,
or proteins) act as a charged species in the formation of PCM
[8]. As a result, PCM self-assemble into a nanoscale core-corona
structure with ionic segment-drug complex as the core and the
water-soluble nonionic segments (PEG) as the outer corona. The
neutral polymer segment, which forms the corona of the PCM,
ensures aqueous solubility under stoichiometric conditions. Furthermore, the hydrophilic shell prevents the aggregation and phase
separation of micelles, and improves their stability [9].
It is well known that stability of electrostatically assembled PCM
relies on the nature and charge density of ionic species involved.
The nature of the interaction determines its binding strength. The
stronger the charge interaction between the ionic segments, the
stronger and more stable will the micellar complex be, which lays
the foundation for its in vivo stability. Obtaining in vivo stability
represents a challenge for the successful delivery of nanoparticles
to tumor interstitial spaces [10].
Well-known anticancer drugs doxorubicin (DOX) and mitoxantrone (MTX) are anthracyclines with a broad spectrum of activity
against a variety of cancers including breast, lung, prostate, bone,
and bladder cancers. These anticancer agents act by intercalating
DNA and inhibiting topoisomerase II [11]. While both DOX and MTX
are anthracycline moieties, they differ in number and substitution
states of amino functionalities present. DOX consists of four fused
rings with a sugar moiety containing a primary amine group (-NH2 ),
while MTX has three fused rings containing two secondary amine
groups (-NH-) [6,11]. Both DOX and MTX are positively charged at
physiological pH with an average pKa of ∼ 8, which is responsible
for electrostatic interactions with the carboxylate group (pKa ∼ 5)
of the block copolymer [12]. In the present study, poly(ethylene
oxide)-block-poly(acrylic acid) (PEO-b-PAA) was used as the block
copolymer. The PCM were formed by the electrostatic interaction of
the protonated amino groups of MTX or DOX with the carboxylate
moiety of the PAA segment of the PEO-b-PAA polymer.
The present study aimed to investigate the interactions of different cationic drugs with the anionic block polymer PEO-b-PAA and
to study the influence of these interactions on the pharmacokinetic
stability and antitumor potential of the formed PCM in clinically
relevant animal models. Towards this purpose, pH-responsiveness
and release profiles of individual drugs from drug-loaded PCM were
monitored. In addition, an in vivo pharmacokinetic study of PCM
(DOX-PCM and MTX-PCM) in rats and an antitumor efficacy study
in A-549 cancer cell-xenografted mouse models were performed.
The effect of amine-functionalized anticancer drugs on the physicochemical and biological responses of micellar nanocarriers was
demonstrated.
2. Materials and methods
2.1. Materials
Doxorubicin hydrochloride was supplied by Dong-A Pharmaceutical Company (Yongin, South Korea). Mitoxantrone dihydrochloride was purchased from Shaanxi Top Pharm Chemical
Co. Ltd (Xi’an, China). Poly(ethylene oxide)-block-poly(acrylic acid)
(PEO-b-PAA, MWs of PEO and PAA blocks were 5000 and 6800 Da,
respectively) was procured from Polymer Source, Inc. (Quebec,
Canada). All other chemicals were of reagent grade purity and were
used without any further modifications.
2.2. Preparation of drug-loaded PCM
The MTX and DOX-loaded PCM were formed by a simple mixing
method as we reported previously [6]. Briefly, aqueous solutions

153

of the polymer (PEO-b-PAA) and drug (MTX or DOX) were prepared separately and mixed together at various charge ratios
of amino groups of drugs to carboxylate groups of the polymer
(R = [drug]/[COO− ]). The mixture was incubated at 25 ◦ C for 24 h
during which the drug and the polymer block self-assembled to
form core-shell micelles. The pH of the solution mixture was
changed to investigate the effect of pH on the degree of ionization and micelle forming. The free unbound drugs were removed
thoroughly by repeated filtrations using Amicon YM-10 centrifugal filter devices (MWCO, 10000 Da; Millipore, Billerica, MA, USA)
pretreated with drugs to retain only the drug-loaded micelles. The
concentration of MTX or DOX in the filtrate was estimated by
UV/VIS spectrophotometry (Perkin Elmer U-2800, Hitachi, Japan).
The wavelengths of 609 and 485 nm were selected for measuring
MTX and DOX, respectively.
2.3. Particle size and ␨-potential analysis
Particle size (nm), polydispersity index (PDI), and zeta (␨)potential (mV) of MTX-PCM and DOX-PCM were analyzed by
dynamic light scattering (DLS). Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) equipped with He–Ne laser was used to
measure the particle size. A fixed angle of 90◦ was selected and the
laser was operated at 635 nm. Nano DTS software (version 6.34)
was employed to analyze the size, PDI, and surface charge of the
micelles. Each measurement was performed in triplicate.
2.4. Morphological analysis
Transmission electron microscopy (TEM) (CM 200 UT; Philips,
Andover, MA, USA) was used to characterize the morphology of
drug-loaded PCM. The particles were observed at an accelerating
voltage of 100 kV. Briefly, a drop of micellar dispersion (R = 0.5) was
placed in the carbon-coated copper grid and allowed to settle for
10 min. Excess liquid was soaked out with tissue paper. The thin
layer of particles was counter-stained by 2% phosphotungstic acid
(PTA) as a negative staining. The particles were subjected to infrared
radiation for 5 min.
2.5. Physical state characterization
The X-ray diffraction (XRD) patterns of free DOX, MTX, DOXPCM, and MTX-PCM were recorded using a vertical goniometer and
X-ray diffractometer (X’Pert PRO MPD diffractometer, Almelo, The
Netherlands) to measure Ni-filtered CuK␣ radiation (voltage, 40 kV;
current, 30 mA) scattered in the crystalline regions of the sample.
The patterns were recorded at a scanning rate of 5◦ /min over the
10–60◦ diffraction angle (2 ␪) range at an ambient temperature.
2.6. In vitro release studies
The release profiles of drugs from MTX-PCM or DOX-PCM
were evaluated by dialysis. Phosphate-buffered saline (PBS, pH 7.4,
0.14 M NaCl) and acetate-buffered saline (pH 5.0, 0.14 M NaCl) were
used to simulate the physiological and tumor pH. In brief, 1 ml of
micellar dispersion (1 mg equivalent of MTX and DOX at R = 0.5)
was sealed in membrane tubing (Spectra/Por® ; 3500 Da cutoff) and
placed at 37 ◦ C at 100 rpm. The samples were withdrawn at predetermined times and replaced with equal amounts of fresh medium.
The samples were collected, filtered, and analyzed using UV–vis
spectrophotometry at 609 and 485 nm for MTX and DOX, respectively. The amount of drug released was plotted against time. The
release kinetics was analyzed by fitting the data to appropriate
mathematical models.


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Fig. 1. Schemes illustrating preparation of drug-loaded PCM.

2.7. Cell culture
A-549 small lung cancer cells were grown in RPMI 1640 medium
supplemented with 10% (v/v) fetal bovine serum (FBS) in the presence of penicillin and streptomycin (100 U/mL and 0.1 mg/mL,
respectively). The cells were maintained under ambient conditions
(37 ◦ C containing 5% CO2 ) in a T-75 flask and periodically subcultured.
2.8. Cellular uptake analysis
The cellular uptake of free drugs and drug-loaded PCM was
investigated in A-549 cancer cells using fluorescence-assisted cell
sorting (FACS). Briefly, cells were seeded at a density of 3 × 105
cells/well in a 6-well plate and incubated overnight. The cells were
treated with free DOX, free MTX, DOX-PCM, and MTX-PCM (in
equivalent concentrations of 10 ␮g/mL) and incubated for the indicated periods. The cells were washed twice with PBS and harvested.
The cells were resuspended in 1 mL of PBS and analyzed in a flow
cytometer (FACSCalibur, BD Biosciences, San Jose, CA, USA).
2.9. Apoptosis analysis
The cells were seeded at a density of 3 × 105 cells/well in a 6well plate and incubated overnight. The cells were treated with
free DOX, free MTX, DOX-PCM, and MTX-PCM (in equivalent concentrations of 5 ␮g/mL) and incubated for 24 h. Next day, cells were
washed, trypsinized, harvested, and washed again with cold PBS.
The pellet was treated with 2.5 ␮L of Annexin V-FITC and 2.5 ␮L of
7-AAD for 15 min at room temperature. The percentage of apoptotic cells was analyzed using a flow cytometer (FACSCalibur, BD
Biosciences, San Jose, CA, USA).

Korea. The rats were divided into four groups with 4 rats in each
group.
2.11. Administration and blood collection
The rats were held in a supine position. The right femoral artery
was cannulated to collect the blood samples, while the left femoral
artery was cannulated to administer the individual DOX and MTX
formulations as a single dose (5 mg/kg). 300 ␮L of prepared PCM
formulations were administered to each rat via a tail vein injection. The micelles formulated at a feeding ratio of R = 0.5 were
employed. Blood samples (200 ␮L) were collected at designated
intervals (0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h). The surgical openings were immediately sealed with surgical sutures to ease pain and
increase the length of the study period. After blood was withdrawn,
it was immediately centrifuged (Eppendorf, Hauppauge, NY, USA)
at 13 000 rpm for 10 min so that plasma could be separated and
extracted for further analysis.
2.12. Preparation and evaluation of plasma samples by HPLC
150 ␮L of plasma was mixed with 150 ␮L of methanol and
vortex-mixed for 30 min. The mixture was centrifuged at high
speed; supernatant was separated and subjected to vacuum evaporation. The evaporated residue was reconstituted with mobile
phase and injected into the HPLC column (20 ␮L). Two different mobile phases were used: sodium formate (80 nM)/methanol
(80/20; pH 2.9) for MTX and methanol/water/acetic acid (50/49/1;
pH 3) for DOX. Flow rate of the mobile phase was 1 ml/min and
effluents were measured at 254 and 480 nm for MTX and DOX,
respectively.
2.13. Pharmacokinetic parameters

2.10. Pharmacokinetic analysis
The in vivo pharmacokinetic study was performed in male
Sprague-Dawley rats (220 ± 10 g). The experimental protocols and
animal care were in accordance with the protocols laid by Institutional Animal Ethical Committee, Yeungnam University, South

A non-compartmental model was used to plot the plasma
concentration–time values using WinNonlin software (professional
edition, version 2.1; Pharsight Corporation, Mountain View, CA,
USA). Pharmacokinetic parameters included the elimination rate
(Kel ), half-life (t1/2 ), maximum plasma concentration (Cmax ), time


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155

of Cmax (Tmax ), mean retention time (MRT) and area under the
plasma concentration–time curve (AUC).
2.14. In vivo antitumor efficacy study
The experimental protocols and animal care were in accordance
with the protocols laid by Institutional Animal Ethical Committee,
Yeungnam University, South Korea. Antitumor efficacy of formulations was studied on 7-week old female BALB/c nude mice.
The mice were maintained on 12-h daylight cycle with access
to food and water. The A-549 cell suspension (5 × 106 cells in
0.1 mL PBS) was subcutaneously injected into the right flanks of
the mice. Prior to the study, the mice were equally divided into five
groups with 6 mice in each group. Mice in four groups received
free MTX, free DOX, MTX-PCM, and DOX-PCM respectively, at a
fixed dose of 5 mg/kg. Each mouse received 100 ␮L of the formulation administered. The untreated group was observed as control.
After the appropriate tumor volume was attained, formulations
were injected via tail vein (3 times with a gap of 3 days between
each injection). The tumor volume was measured (V = 0.5 × longest
diameter × shortest diameter) using a Vernier caliper. The body
weight of mice was monitored in order to observe the safety profile of the formulations. At the end of the experiment, mice were
sacrificed according to the institutional ethical guidelines. Tumors
were surgically removed and weighed individually.
2.15. Statistical analysis
Student’s t-test was used to evaluate the statistical significance of differences between formulations. Values were reported
as mean ± standard deviation (SD) and the data were considered
statistically significant at p < 0.05.
3. Results and discussion
3.1. Preparation of drug-loaded PCM
The present study aimed at investigating the interactions of different cationic drugs with the anionic block polymer PEO-b-PAA.
The primary amino group of DOX (-NH2 ) and two secondary amino
groups (-NH-) of MTX are responsible for electrostatic interactions
with the ionized carboxyl group (pKa ∼ 5) of PEO-b-PAA [11]. Therefore, pH-responsiveness of individual drugs and their ability to form
drug-loaded PCM were studied in detail. The PCM were formed at
two different charge ratios (R = 0.25 and 0.5) of MTX and DOX to
carboxylate groups (R = [drug]/[COO− ]). The schematic illustration
of formation of PCM is presented in Fig. 1.
As shown in Fig. 2, the PCM were prepared with both drugs at
various pH conditions. The PCM were formed by the immobilization
of weakly basic drugs (MTX and DOX) into the cores of PEO-b-PAA,
a weak polyacid, via a strong electrostatic interaction. As expected,
we observed pH-sensitive behavior of PCM. Compared to PCM particle size at pH 6, particle size reduced remarkably with the increase
in pH (DOX-PCM). Consistently, ␨-potential of PCM decreased as
pH increased, indicating that higher pH favors the ionization of the
polymer block resulting in complexation of drugs. The ␨-potential
decreased in the entire pH range studied. A possible interpretation
is that at lower pH values, owing to partial or insufficient ionization of the PAA block, the drug-polymer physical interaction forms
a loose aggregate resulting in larger particle size. Upon increase in
the pH of the medium, PAA attains maximum ionization resulting
in efficient complexation of drugs. It is worth noting that particle
size markedly decreased when the charge ratio was increased from
R = 0.25 to R = 0.5, indicating the neutralization of the PAA segments
due to the electrostatic interaction of MTX and DOX in the PCM. As
expected at higher charge ratios, greater neutralization of negative

Fig. 2. Effect of pH on (A) hydrodynamic particle size and (B) ␨-potential at different
feeding ratios (R = 0.25 and 0.5).

charge of the PAA block results in stable PCM with highly hydrophobic core and high payload capacity [13]. Appreciable hydrophobic
core and PEG shell on the surface stabilize the PCM in systemic
conditions. Specifically, MTX-PCM exhibited a smaller particle size
compared to that of DOX-PCM. This difference in particle sizes of
cationic drug-based PCM can be attributed to the binding strength
of individual drugs to the polymer block. Overall, the size of MTXPCM and DOX-PCM was less than 100 nm, making these PCM ideal
for tumor drug delivery. It has been reported that particle smaller
than 200 nm can preferentially accumulate in tumor tissues via
diffusion-mediated passive transport (EPR effect), whereas particles smaller than 100 nm can penetrate deep in the leaky tumor
vasculature (typical pore size 50–100 nm) and are not limited to
vascular surface only [14,15].


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Fig. 3. TEM images of (A) MTX-PCM and (B) DOX-PCM.

3.2. Morphological and physical state analysis
Regardless of the nature of binding of individual drugs, both
MTX-PCM and DOX-PCM exhibited distinct spherical shaped particles uniformly dispersed on the TEM grid (Fig. 3A, B). Core-shell
architecture was not observed due to the longitudinal assembly
of polymer chains, but an electron-dense dark core representing
the drug-polymer complex was seen. The particle size observed
in the TEM experiment was smaller than size observed using DLS.
This discrepancy in observed sizes can be attributed to the fact that
DLS measures the hydrodynamic micelle size while TEM captures
the dried state. The physical state of free drugs and drug-loaded
micelles was studied using X-ray diffraction patterns. As shown in
Fig. S1, free DOX showed numerous sharp and intense peaks at sev◦





eral 2 ␪ scattered angles (12.5 , 16.2 , 17.3 , 21.2 , 22.5 , 25.1 , and

26.2 ) and free MTX showed peaks between 22.5 − 25.5◦ reflecting
its high crystallinity. All characteristic peaks were absent in DOXPCM as well as MTX-PCM, indicating the complete incorporation of
drugs. These results suggest the presence of drugs in the amorphous
or molecularly dispersed state [16].

3.3. Drug loading and in vitro release study
Both the DOX-PCM and MTX-PCM exhibited a high entrapment
efficiency of more than 90% with an active drug loading of ∼ 45%
w/w for MTX (MTX-PCM) and ∼ 70% for DOX (DOX-PCM) at R = 0.5.
The release study of MTX and DOX from MTX-PCM and DOX-PCM
was performed in phosphate-buffered saline (pH 7.4) and acetatebuffered saline (pH 5.0) to simulate the physiological and tumor
pH conditions. As evident from Fig. 4, MTX-PCM and DOX-PCM
exhibited different release profiles at both pH conditions. MTXPCM exhibited a sustained release profile throughout the study
period with no initial burst. DOX-PCM, on the other hand, exhibited
a biphasic release pattern. DOX-PCM exhibited a faster release profile during the initial time interval (10–12 h), but showed a slower
release later on (48 h). For instance, in the case of R = 0.5, ∼ 9% of
the drug was released from MTX-PCM after 12 h and approximately
25% of the drug was released by the end of 48 h at pH 7.4. In contrast, ∼ 30% of the drug was released from DOX-PCM during the
first 12 h, while the total release was ∼ 38% at the end of the study
period. A similar trend was observed in release media at pH 5.0
wherein MTX was released in a continuous fashion (monophasic), while DOX was released in a biphasic manner. The difference
in release patterns could be attributed to the charge density and
binding affinity of individual drugs towards the anionic PAA block.

Fig. 4. Release profiles of (A) MTX and (B) DOX from MTX-PCM and DOX-PCM at
pH 5.0 and pH 7.4. MTX-PCM and DOX-PCM were prepared at pH 7.0. The study was
carried out in phosphate-buffered saline (pH 7.4) and acetate-buffered saline (pH
5.0) at 37 ◦ C.


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Fig. 5. (A) In vitro cellular uptake of (a) MTX and MTX-PCM and (b) DOX and DOX-PCM in SCC-7 cancer cells. The cellular uptake experiment was performed by incubating
the formulations at different time points. (B) Annexin V-FITC/PI based apoptosis assay in SCC-7 cancer cells. The drug treated cells were stained with Annexin V-FITC and PI
and evaluated using flow cytometry.

MTX, with two secondary amino groups could be expected to have
stronger binding affinity for the polymer than DOX with a single
primary amino group.
Another significant observation was that MTX and DOX were
released faster in acidic pH (pH 5.0) than in physiological pH (pH
7.4). For example, approximately 55% of MTX was released from
MTX-PCM at acidic pH, while only ∼ 25% of the drug was released
at physiological pH after 48 h at a feeding ratio of R = 0.5. A similar
trend was observed in the case of DOX-PCM, wherein ∼ 62% of the
drug was released in acidic media comparing to ∼ 38% DOX release
after 48 h in basic media. The accelerated release of drugs at acidic
pH can be attributed to the protonation of carboxylic groups of the
PAA block in the micelles [11].
In general, it is interesting to note that the release rate was
higher from PCM prepared at a feeding ratio of R = 0.5 than from
those prepared with R = 0.25. For example, ∼ 14% of MTX was
released from MTX-PCM with R = 0.25 and ∼ 21% was released from
MTX-PCM with R = 0.5 during the first 8 h at pH 5.0. A similar trend
was observed at pH 7.4: ∼ 17% of DOX was released from DOX-PCM
with R = 0.25 and ∼ 23% was released from PCM with R = 0.5 during the first 8 h. This difference in release can be attributed to the
binding and localization of the drugs in the core of PCM. A high
loading capacity of PCM at R = 0.5 accounts for the larger presence
of drugs at the core-shell interface from where the drugs can rapidly
diffuse into the release medium [17]. The greater number of drug

molecules trapped in the core of PCM prepared with R = 0.5 has a
greater chance to release quickly in the media than the fewer drug
molecules from the PCM prepared with R = 0.25. Furthermore, at
the low feeding ratio (R = 0.25), considerable negative charges are
still available on the PAA chain that will further induce strong electrostatic interactions between drugs and the polymer leading to
slower release rates.

3.4. Cellular uptake patterns of DOX-PCM and MTX-PCM
The cellular uptake behavior of free drugs and drug-loaded PCM
was investigated in SCC-7 cancer cells using FACS [18,19]. As shown
in Fig. 5A, free DOX and MTX showed a higher cellular uptake compared to drug-loaded PCM. The higher cellular uptake of free drugs
was attributed to the simple diffusion of drugs to the intracellular
environment, whereas micellar nanocarriers could only be internalized by the cells through endocytosis. The mean fluorescent
intensity (MFI) of free DOX was greater compared to DOX-PCM and
similarly, MFI of MTX was greater compared to that of MTX-PCM
after 60-min incubation in SCC-7 cancer cells. Consistently, DOXPCM and MTX-PCM showed a typical time-dependent behavior due
to the presence of an endocytosis process within the system (Fig.
S2). We have observed that the nanocarriers primarily accumulate in the cytoplasmic region where the drug is liberated after the


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degradation of delivery systems. These results indicate an efficient
internalization of PCM in this cell line.

3.5. Apoptosis assay
The externalization of phosphatidylserine during apoptosis of
cancer cells was evaluated by annexin-V/PI staining (Fig. 5B). The
results showed that DOX induced a significant increase in Annexin
V-positive and Annexin V + PI-positive cells, corresponding to
early and late apoptotic cells, respectively. The MTX, however,
induced apoptosis as well as necrosis of cancer cells. Importantly,
drug-loaded PCM induced higher apoptosis rates of cancer cells
compared to free drugs. For example, MTX-PCM induced ∼ 25%
of cell apoptosis compared to ∼ 15% induced by free MTX. Similarly, DOX-PCM induced ∼ 34% of cell apoptosis compared to ∼ 25%
induced by free DOX. Higher cellular apoptosis rates induced by
PCM could be attributed to the sustained release of therapeutic
cargo in the intracellular environment. It should be noted that DOXbased therapy was more effective in inducing anticancer activity
than MTX-based therapy.

3.6. Pharmacokinetic analysis
The plasma concentration-time profiles of free drugs, MTX-PCM,
and DOX-PCM following single dose administration are presented
in Fig. 6. As shown, free MTX and free DOX were cleared from
the systemic compartment within 4–6 h of intravenous administration. Linear pharmacokinetic profiles of MTX and DOX were
consistent with previous reports. As expected, PCM formulations
significantly enhanced the blood circulation of both MTX and DOX.
Both anticancer drugs maintained significantly higher plasma concentrations for 24 h. Importantly, MTX-PCM showed prolonged
blood circulation, compared to DOX-PCM. After 12 h, the plasma
concentration of MTX from PCM was 1.824 ± 0.801 ␮g/ml compared to plasma concentration of only 0.576 ± 0.389 ␮g/mL for
DOX. The final concentrations of MTX and DOX released from PCM
were 1.258 ± 0.392 ␮g/mL and 0.176 ± 0.151 ␮g/mL, respectively.
The respective pharmacokinetic parameters of different formulations are presented in Table 1. Consistent with previous reports,
free drugs exhibited short t1/2 , high Kel , and low AUCall . Although
both PCM formulations improved the systemic performance of
drugs, they markedly differ among themselves. For instance, MTXPCM improved the AUCall of MTX 4-fold compared to a 3-fold
increase by DOX-PCM for DOX. Similarly, MTX-PCM had an approximately 5-fold (14.79 ± 4.89 h) higher t1/2 than MTX, compared with
2.5-fold higher t1/2 of DOX-PCM (4.82 ± 0.83 h) in relation to DOX.
Notably, Kel of the free drugs was reduced 5-fold by MTX-PCM and
2-fold by DOX-PCM. With regard to all pharmacokinetic parameters, MTX-PCM showed 2-fold higher performance than DOX-PCM.
These findings indicate the remarkable blood circulation potential
of MTX-PCM compared to that of DOX-PCM. The difference in the
circulatory performance of the two PCM formulations is attributed
to their physiological stability [20]. Previously, we have shown that
DOX-PCM have lower salt stability than MTX-PCM. Two secondary
amino groups confer on MTX a stronger binding affinity for the
polymer, compared to DOX with its single primary amino group
[11,21].
Many inferences can be drawn from this experiment. First, the
binding affinity of the cationic drug to the polymer determines its
blood circulation potential; second, based on the binding strength,
the release of the drug will be sustained or faster; third, the greater
the binding strength, the greater the in vivo performance of drugloaded nanocarriers in the physiological environment [22–25].

Fig. 6. Plasma concentration-time profiles of MTX and DOX after intravenous
administration of free drugs or drug-loaded PCM to rats at a dose of 5 mg/kg. Each
value represents the mean ± SD (n = 4). Drug-loaded PCM were prepared at R = 0.5
and pH 7.0.

3.7. In vivo antitumor efficacy
The prolonged blood circulation and controlled release profiles of drug-loaded PCM were expected to contribute to their
superior antitumor efficacy. The antitumor efficacy of individual
formulations was investigated in A-549 cancer cells xenografted
on BALB/c nude mice. Free MTX, free DOX, MTX-PCM, and DOXPCM were intravenously injected into the tumor bearing mice at
a fixed dose of 5 mg/kg. As shown in Fig. 7A, tumors rapidly grew
in the untreated control group, but their growth was significantly
suppressed in groups treated with free drugs as well as drugloaded PCM. Notably, both MTX-PCM and DOX-PCM significantly
suppressed tumor growth. In vitro cytotoxicity assays revealed the
IC50 values for individual formulations. The IC50 values of MTX and
MTX-PCM were found to be 0.85 ␮g/ml and 0.94 ␮g/ml, respec-


T. Ramasamy et al. / Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160

159

Table 1
Pharmacokinetic parameters of MTX and DOX after IV administration of free drug or drug-loaded PCM to rats.
Parameters
Kel (h−1 )
t1/2 (h)
AUCall (h ␮g ml−1 )
AUCinf (h ␮g ml−1 )
Cl (␮g ml−1 h−1 )
AUMC (␮g ml−1 h)
MRT (h)

MTX
0.25
2.88
13.18
14.42
141.87
42.78
3.23

±
±
±
±
±
±
±

0.049
0.514
1.497
2.325
20.59
6.34
0.135

MTX-PCM
0.052
14.79
47.85
78.32
31.94
309.71
9.58

±
±
±
±
±
±
±

DOX

0.016
4.895
14.82
35.22
15.12
257.49
0.615

0.36
1.93
8.18
8.72
237.44
18.66
2.26

±
±
±
±
±
±
±

0.054
0.325
1.27
1.72
41.32
3.78
0.118

DOX-PCM
0.15
4.82
27.66
29.03
79.82
162.82
5.71

±
±
±
±
±
±
±

0.022
0.823
8.85
9.78
32.92
67.68
0.72

results show that despite the difference in circulatory performance
and drug release patterns between MTX-PCM and DOX-PCM their
therapeutic effect is similar. It should be noted that DOX-PCM were
more effective than free DOX in suppressing tumors, whereas antitumor efficacy of MTX-PCM and free MTX was observed to be
similar. This was attributed to the high toxicity of MTX on mice,
which resulted in overall body weakness that affected the tumor
tissue as well. The enhanced tumor regression caused by PCM formulations can be attributed to the prolonged half-life of anticancer
drugs, reduced elimination of individual drugs, and most importantly to the preferential accumulation of nanocarriers in the tumor
tissue due to the EPR effect [26–28].
The toxicity of formulations was evaluated using mice body
weight (Fig. 7B). As shown, free MTX caused an approximately 30%
decrease in body weight indicating its severe drug-related toxicity.
MTX-PCM, however, greatly reduced MTX toxicity in systemic circulation. This could be due to the fact that encapsulation of MTX in
the PCM reduced the random exposure of normal tissues to it and
increased MTX’s passive accumulation in tumor tissues, thereby
reducing the undesirable side effects [29,30]. DOX-PCM did not
exhibit any body weight reduction.
4. Conclusion

Fig. 7. Effect of drug-loaded PCM on (A) tumor growth and (B) body weight in A-549
xenograft-bearing female BALB/c nude mice (n = 6 per group). Each formulation was
administered three times at three day intervals. Drug-loaded PCM were prepared at
R = 0.5 and pH 7.0.

tively, while the IC50 values of DOX and DOX-PCM were 1.68 ␮g/ml
and 2.13 ␮g/ml, respectively. While a definite difference in blood
circulation was observed between MTX-PCM and DOX-PCM in the
pharmacokinetic study, no significant difference in antitumor efficacy could be detected. Both MTX-PCM and DOX-PCM inhibited
tumor growth to the same level throughout the study period. These

In summary, cationic drugs-loaded PCM were prepared and
evaluated in terms of physicochemical and in vivo parameters. Both
MTX-PCM and DOX-PCM displayed spherical nanosized particles
with uniform dispersity indices. MTX-PCM and DOX-PCM exhibited
different release profiles under all pH conditions studied. MTX-PCM
exhibited a monophasic release pattern with no initial burst, while
DOX-PCM exhibited a biphasic release pattern. Interestingly, drug
release rates were higher from PCM prepared at a feeding ratio of
R = 0.5 than from those prepared with R = 0.25. DOX-PCM showed
a higher cellular uptake compared to MTX-PCM in SCC-7 cancer
cells; consistently DOX-PCM induced higher apoptosis rates of cancer cells than MTX-PCM. In contrast, MTX-PCM showed prolonged
blood circulation compared to DOX-PCM. MTX-PCM improved the
AUCall of MTX 4-fold compared to a 3-fold increase by DOX-PCM for
DOX. Similarly, MTX-PCM had a 5-fold higher t1/2 than MTX, while
DOX-PCM increased the DOX t1/2 2.5-fold. However, both MTXPCM and DOX-PCM suppressed tumor growth to the same levels as
their respective free drugs. Taken together, our results show that
nature of interactions of cationic drugs with the polyionic copolymer can have a tremendous influence on the biological performance
of delivery systems.
Conflict of interests
The authors declare no conflict of interest in this work.
Acknowledgements
This work was supported by the 2015 Yeungnam University
Research Grant.


160

T. Ramasamy et al. / Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160

Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.06.
004.
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