Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160 Contents lists available at ScienceDirect 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 June 2016 Available online 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 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 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 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 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, 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 154 T Ramasamy et al / Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160 Fig 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 × 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 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 × 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 ␮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 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 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 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 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 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 T Ramasamy et al / Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160 155 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 mice in each group Mice in four groups received free MTX, free DOX, MTX-PCM, and DOX-PCM respectively, at a fixed dose of 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 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 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 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 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] 156 T Ramasamy et al / Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160 Fig 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 ␪ 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 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 T Ramasamy et al / Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160 157 Fig (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 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 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 158 T Ramasamy et al / Colloids and Surfaces B: Biointerfaces 146 (2016) 152–160 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 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 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 Plasma concentration-time profiles of MTX and DOX after intravenous administration of free drugs or drug-loaded PCM to rats at a dose of 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 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 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 Conclusion Fig Effect of drug-loaded PCM on (A) tumor growth and (B) body weight in A-549 xenograft-bearing female BALB/c nude mice (n = 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 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intercalating DNA and inhibiting topoisomerase II [11] While both DOX and MTX are anthracycline moieties, they differ in number and substitution states