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Article history: Nanofabrication of polymeric micelles through self-assembly of an ionic block copolymer and oppositely
Received 22 March 2016 charged small molecules has recently emerged as a promising method of formulating delivery systems.
Received in revised form 31 May 2016 The present study therefore aimed to investigate the interaction of cationic drugs doxorubicin (DOX)
Accepted 2 June 2016
and mitoxantrone (MTX) with the anionic block polymer poly(ethylene oxide)-block-poly(acrylic acid)
Available online 5 June 2016
(PEO-b-PAA) and to study the influence of these interactions on the pharmacokinetic stability and anti-
tumor potential of the formulated micelles in clinically relevant animal models. To this end, individual
Keywords:
DOX and MTX-loaded polyelectrolyte complex micelles (PCM) were prepared, and their physicochemical
Nanofabrication
Polyelectrolyte complex micelles properties and pH-responsive release profiles were studied. MTX-PCM and DOX-PCM exhibited a differ-
Cationic drugs ent release profile under all pH conditions tested. MTX-PCM exhibited a monophasic release profile with
Pharmacokinetic no initial burst, while DOX-PCM exhibited a biphasic release. DOX-PCM showed a higher cellular uptake
Anticancer activity 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 pro-
longed 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 tic drugs, various drug delivery systems have been developed.
Among them, block copolymer-based self-assembled polymeric
Conventional chemotherapeutic approach is the main treat- micelles have demonstrated promising potential in the delivery
ment option for cancer [1]. Despite great strides made in of anticancer drugs. The nanosized micelles offer many advan-
understanding cancer biology, conventional chemotherapeutic tages, including uniform size distribution, core-shell architecture,
drugs are characterized by non-specific distribution and high accu- high drug loading, and physical stability [3,4]. Polyethylene glycol
mulation in healthy cells, leading to dose-limiting side effects (PEG) is widely used to graft the hydrophobic part of amphiphilic
that seriously impede their clinical application [2]. To minimize polymers and form the outer shell of the micelles. Such polymeric
side effects and improve therapeutic efficacy of chemotherapeu- 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
∗ Corresponding authors. micelles formed by electrostatic interaction of opposite charged
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.
T. Ramasamy et al. / Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160 153
species (ionic blocks), have recently been developed [6,7]. For drug of the polymer (PEO-b-PAA) and drug (MTX or DOX) were pre-
delivery applications, therapeutic moieties (small molecules, DNA, pared separately and mixed together at various charge ratios
or proteins) act as a charged species in the formation of PCM of amino groups of drugs to carboxylate groups of the polymer
[8]. As a result, PCM self-assemble into a nanoscale core-corona (R = [drug]/[COO− ]). The mixture was incubated at 25 ◦ C for 24 h
structure with ionic segment-drug complex as the core and the during which the drug and the polymer block self-assembled to
water-soluble nonionic segments (PEG) as the outer corona. The form core-shell micelles. The pH of the solution mixture was
neutral polymer segment, which forms the corona of the PCM, changed to investigate the effect of pH on the degree of ioniza-
ensures aqueous solubility under stoichiometric conditions. Fur- tion and micelle forming. The free unbound drugs were removed
thermore, the hydrophilic shell prevents the aggregation and phase thoroughly by repeated filtrations using Amicon YM-10 centrifu-
separation of micelles, and improves their stability [9]. gal filter devices (MWCO, 10000 Da; Millipore, Billerica, MA, USA)
It is well known that stability of electrostatically assembled PCM pretreated with drugs to retain only the drug-loaded micelles. The
relies on the nature and charge density of ionic species involved. concentration of MTX or DOX in the filtrate was estimated by
The nature of the interaction determines its binding strength. The UV/VIS spectrophotometry (Perkin Elmer U-2800, Hitachi, Japan).
stronger the charge interaction between the ionic segments, the The wavelengths of 609 and 485 nm were selected for measuring
stronger and more stable will the micellar complex be, which lays MTX and DOX, respectively.
the foundation for its in vivo stability. Obtaining in vivo stability
represents a challenge for the successful delivery of nanoparticles
2.3. Particle size and -potential analysis
to tumor interstitial spaces [10].
Well-known anticancer drugs doxorubicin (DOX) and mitox-
Particle size (nm), polydispersity index (PDI), and zeta ()-
antrone (MTX) are anthracyclines with a broad spectrum of activity
potential (mV) of MTX-PCM and DOX-PCM were analyzed by
against a variety of cancers including breast, lung, prostate, bone,
dynamic light scattering (DLS). Zetasizer Nano ZS (Malvern Instru-
and bladder cancers. These anticancer agents act by intercalating
ments, Malvern, UK) equipped with He–Ne laser was used to
DNA and inhibiting topoisomerase II [11]. While both DOX and MTX
measure the particle size. A fixed angle of 90◦ was selected and the
are anthracycline moieties, they differ in number and substitution
laser was operated at 635 nm. Nano DTS software (version 6.34)
states of amino functionalities present. DOX consists of four fused
was employed to analyze the size, PDI, and surface charge of the
rings with a sugar moiety containing a primary amine group (-NH2 ),
micelles. Each measurement was performed in triplicate.
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 2.4. Morphological analysis
for electrostatic interactions with the carboxylate group (pKa ∼ 5)
of the block copolymer [12]. In the present study, poly(ethylene Transmission electron microscopy (TEM) (CM 200 UT; Philips,
oxide)-block-poly(acrylic acid) (PEO-b-PAA) was used as the block Andover, MA, USA) was used to characterize the morphology of
copolymer. The PCM were formed by the electrostatic interaction of drug-loaded PCM. The particles were observed at an accelerating
the protonated amino groups of MTX or DOX with the carboxylate voltage of 100 kV. Briefly, a drop of micellar dispersion (R = 0.5) was
moiety of the PAA segment of the PEO-b-PAA polymer. placed in the carbon-coated copper grid and allowed to settle for
The present study aimed to investigate the interactions of differ- 10 min. Excess liquid was soaked out with tissue paper. The thin
ent cationic drugs with the anionic block polymer PEO-b-PAA and layer of particles was counter-stained by 2% phosphotungstic acid
to study the influence of these interactions on the pharmacokinetic (PTA) as a negative staining. The particles were subjected to infrared
stability and antitumor potential of the formed PCM in clinically radiation for 5 min.
relevant animal models. Towards this purpose, pH-responsiveness
and release profiles of individual drugs from drug-loaded PCM were
2.5. Physical state characterization
monitored. In addition, an in vivo pharmacokinetic study of PCM
(DOX-PCM and MTX-PCM) in rats and an antitumor efficacy study
The X-ray diffraction (XRD) patterns of free DOX, MTX, DOX-
in A-549 cancer cell-xenografted mouse models were performed.
PCM, and MTX-PCM were recorded using a vertical goniometer and
The effect of amine-functionalized anticancer drugs on the physic-
X-ray diffractometer (X’Pert PRO MPD diffractometer, Almelo, The
ochemical and biological responses of micellar nanocarriers was
Netherlands) to measure Ni-filtered CuK␣ radiation (voltage, 40 kV;
demonstrated.
current, 30 mA) scattered in the crystalline regions of the sample.
The patterns were recorded at a scanning rate of 5◦ /min over the
2. Materials and methods 10–60◦ diffraction angle (2 ) range at an ambient temperature.
2.1. Materials
2.6. In vitro release studies
2.7. Cell culture Korea. The rats were divided into four groups with 4 rats in each
group.
A-549 small lung cancer cells were grown in RPMI 1640 medium
supplemented with 10% (v/v) fetal bovine serum (FBS) in the pres-
2.11. Administration and blood collection
ence of penicillin and streptomycin (100 U/mL and 0.1 mg/mL,
respectively). The cells were maintained under ambient conditions
The rats were held in a supine position. The right femoral artery
(37 ◦ C containing 5% CO2 ) in a T-75 flask and periodically sub-
was cannulated to collect the blood samples, while the left femoral
cultured.
artery was cannulated to administer the individual DOX and MTX
formulations as a single dose (5 mg/kg). 300 L of prepared PCM
2.8. Cellular uptake analysis formulations were administered to each rat via a tail vein injec-
tion. The micelles formulated at a feeding ratio of R = 0.5 were
The cellular uptake of free drugs and drug-loaded PCM was employed. Blood samples (200 L) were collected at designated
investigated in A-549 cancer cells using fluorescence-assisted cell intervals (0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h). The surgical open-
sorting (FACS). Briefly, cells were seeded at a density of 3 × 105 ings were immediately sealed with surgical sutures to ease pain and
cells/well in a 6-well plate and incubated overnight. The cells were increase the length of the study period. After blood was withdrawn,
treated with free DOX, free MTX, DOX-PCM, and MTX-PCM (in it was immediately centrifuged (Eppendorf, Hauppauge, NY, USA)
equivalent concentrations of 10 g/mL) and incubated for the indi- at 13 000 rpm for 10 min so that plasma could be separated and
cated periods. The cells were washed twice with PBS and harvested. extracted for further analysis.
The cells were resuspended in 1 mL of PBS and analyzed in a flow
cytometer (FACSCalibur, BD Biosciences, San Jose, CA, USA).
2.12. Preparation and evaluation of plasma samples by HPLC
2.9. Apoptosis analysis 150 L of plasma was mixed with 150 L of methanol and
vortex-mixed for 30 min. The mixture was centrifuged at high
The cells were seeded at a density of 3 × 105 cells/well in a 6- speed; supernatant was separated and subjected to vacuum evap-
well plate and incubated overnight. The cells were treated with oration. The evaporated residue was reconstituted with mobile
free DOX, free MTX, DOX-PCM, and MTX-PCM (in equivalent con- phase and injected into the HPLC column (20 L). Two differ-
centrations of 5 g/mL) and incubated for 24 h. Next day, cells were ent mobile phases were used: sodium formate (80 nM)/methanol
washed, trypsinized, harvested, and washed again with cold PBS. (80/20; pH 2.9) for MTX and methanol/water/acetic acid (50/49/1;
The pellet was treated with 2.5 L of Annexin V-FITC and 2.5 L of pH 3) for DOX. Flow rate of the mobile phase was 1 ml/min and
7-AAD for 15 min at room temperature. The percentage of apop- effluents were measured at 254 and 480 nm for MTX and DOX,
totic cells was analyzed using a flow cytometer (FACSCalibur, BD respectively.
Biosciences, San Jose, CA, USA).
of Cmax (Tmax ), mean retention time (MRT) and area under the
plasma concentration–time curve (AUC).
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 molecules trapped in the core of PCM prepared with R = 0.5 has a
stronger binding affinity for the polymer than DOX with a single greater chance to release quickly in the media than the fewer drug
primary amino group. molecules from the PCM prepared with R = 0.25. Furthermore, at
Another significant observation was that MTX and DOX were the low feeding ratio (R = 0.25), considerable negative charges are
released faster in acidic pH (pH 5.0) than in physiological pH (pH still available on the PAA chain that will further induce strong elec-
7.4). For example, approximately 55% of MTX was released from trostatic interactions between drugs and the polymer leading to
MTX-PCM at acidic pH, while only ∼ 25% of the drug was released slower release rates.
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 3.4. Cellular uptake patterns of DOX-PCM and MTX-PCM
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 The cellular uptake behavior of free drugs and drug-loaded PCM
PAA block in the micelles [11]. was investigated in SCC-7 cancer cells using FACS [18,19]. As shown
In general, it is interesting to note that the release rate was in Fig. 5A, free DOX and MTX showed a higher cellular uptake com-
higher from PCM prepared at a feeding ratio of R = 0.5 than from pared to drug-loaded PCM. The higher cellular uptake of free drugs
those prepared with R = 0.25. For example, ∼ 14% of MTX was was attributed to the simple diffusion of drugs to the intracellular
released from MTX-PCM with R = 0.25 and ∼ 21% was released from environment, whereas micellar nanocarriers could only be inter-
MTX-PCM with R = 0.5 during the first 8 h at pH 5.0. A similar trend nalized by the cells through endocytosis. The mean fluorescent
was observed at pH 7.4: ∼ 17% of DOX was released from DOX-PCM intensity (MFI) of free DOX was greater compared to DOX-PCM and
with R = 0.25 and ∼ 23% was released from PCM with R = 0.5 dur- similarly, MFI of MTX was greater compared to that of MTX-PCM
ing the first 8 h. This difference in release can be attributed to the after 60-min incubation in SCC-7 cancer cells. Consistently, DOX-
binding and localization of the drugs in the core of PCM. A high PCM and MTX-PCM showed a typical time-dependent behavior due
loading capacity of PCM at R = 0.5 accounts for the larger presence to the presence of an endocytosis process within the system (Fig.
of drugs at the core-shell interface from where the drugs can rapidly S2). We have observed that the nanocarriers primarily accumu-
diffuse into the release medium [17]. The greater number of drug late in the cytoplasmic region where the drug is liberated after the
158 T. Ramasamy et al. / Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160
Table 1
Pharmacokinetic parameters of MTX and DOX after IV administration of free drug or drug-loaded PCM to rats.
Kel (h−1 ) 0.25 ± 0.049 0.052 ± 0.016 0.36 ± 0.054 0.15 ± 0.022
t1/2 (h) 2.88 ± 0.514 14.79 ± 4.895 1.93 ± 0.325 4.82 ± 0.823
AUCall (h g ml−1 ) 13.18 ± 1.497 47.85 ± 14.82 8.18 ± 1.27 27.66 ± 8.85
AUCinf (h g ml−1 ) 14.42 ± 2.325 78.32 ± 35.22 8.72 ± 1.72 29.03 ± 9.78
Cl (g ml−1 h−1 ) 141.87 ± 20.59 31.94 ± 15.12 237.44 ± 41.32 79.82 ± 32.92
AUMC (g ml−1 h) 42.78 ± 6.34 309.71 ± 257.49 18.66 ± 3.78 162.82 ± 67.68
MRT (h) 3.23 ± 0.135 9.58 ± 0.615 2.26 ± 0.118 5.71 ± 0.72
4. Conclusion
Appendix A. Supplementary data [16] B. Gupta, B.K. Poudel, S. Pathak, J.W. Tak, H.H. Lee, J.H. Jeong, H.G. Choi, C.S.
Yong, J.O. Kim, Effects of formulation variables on the particle size and drug
encapsulation of imatinib-loaded solid lipid nanoparticles, AAPS
Supplementary data associated with this article can be found, in PharmSciTech (2015).
the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.06. [17] T. Ramasamy, J.H. Kim, J.Y. Choi, T.H. Tran, H.G. Choi, C.S. Yong, J.O. Kim, pH
004. sensitive polyelectrolyte complex micelles for highly effective combination
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[18] X. Qi, Y. Chen, Z. Sun, X. Ma, W. Guo, Y. Lu, Duan, Cytotoxicity and cellular
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