Pharmacokinetics and antitumor efficacy of micelles assembled from multiarmed amphiphilic copolymers with drug conjugates in comparison with drug-encapsulated micelles

European Journal of Pharmaceutics and Biopharmaceutics 98 (2016) 9–19

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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Research Paper

Pharmacokinetics and antitumor efficacy of micelles assembled from multiarmed amphiphilic copolymers with drug conjugates in comparison with drug-encapsulated micelles Xiaoming Luo a,b, Maohua Chen a, Yun Zhang a, Zhoujiang Chen a, Xiaohong Li a,⇑ a b

Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China Department of Public Health, Chengdu Medical College, Chengdu 610500, PR China

a r t i c l e

i n f o

Article history: Received 11 August 2015 Revised 22 October 2015 Accepted in revised form 27 October 2015 Available online 31 October 2015 Keywords: Drug-conjugated micelle Drug-encapsulated micelle Pharmacokinetics Antitumor efficacy Antimetastasis

a b s t r a c t The premature drug release and structural dissociation before reaching pathological sites have posed major challenges for self-assembled micelles. To address these challenges, star-shaped amphiphilic copolymers derived from 4-armed poly(ethylene glycol) (PEG) were proposed for chemical conjugation of chemotherapeutic drugs and assembly into drug-conjugated micelles (DCM) with reductive sensitivity. The current study aimed to elucidate the in vitro and in vivo performance of DCM and a comparison with conventional drug-encapsulated micelles (DEM) was initially launched. DEM carriers were constructed with a similar structure to DCM from 4-armed PEG, and disulfide linkages were located between the hydrophilic and hydrophobic segments. Both DCM and DEM had an average size of around 130 nm, camptothecin (CPT) loadings of around 7.7% and critical micelle concentrations of around 0.95 lg/ml. Compared with DEM, DCM showed a lower initial drug release, a lower sensitivity of drug release to glutathione, and a higher structural stability after incubation with human serum albumin (HSA). The CPT derivatives (CPT-SH) released from DCM indicated cytotoxicities similar to CPT and remained a higher lactone stability than CPT in the presence of HSA. DCM showed slightly higher cytotoxicities to 4T1 cells and significantly lower cytotoxicities to normal cells than DEM. Pharmacokinetic analyses after intravenous administration of DCM indicated around 2.65 folds higher AUC0–1, 2.66 folds lower clearance, and 1.87 folds higher tumor accumulation than those of DEM. In addition to a less disturbance to hematological and biochemical parameters and a lower acute toxicity to small intestines, DCM showed more significant tumor suppression efficacy and less tumor metastasis to lungs than DEM. It is suggested that DCM could overcome the limitation of conventional micelles by alleviating the premature drug release during blood circulation, relieving the systemic toxicity and promoting the therapeutic efficacy. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Chemotherapy is one of the most widely used tools in combating cancer, but current chemotherapeutic agents suffer from several intrinsic limitations, such as poor water solubility, improper biodistribution, and possible occurrence of severe side-effects. In addition to the development of novel anticancer drugs, liposomes, nanoparticles and polymeric micelles have been used to improve the bioavailability, promote the treatment efficacy and reduce the adverse effect of current therapeutic drugs [1]. Currently several products are commercially available in clinical use, and quite a few formulations have been evaluated for different phases of

⇑ Corresponding author. Tel.: +86 28 87634068; fax: +86 28 87634649. E-mail address: [email protected] (X. Li). http://dx.doi.org/10.1016/j.ejpb.2015.10.014 0939-6411/Ó 2015 Elsevier B.V. All rights reserved.

clinical trials [2]. As drug carriers, micelles have manifested several attractive features and advantages over other types of carriers, resulting from the core–shell structure self-assembled from amphiphilic copolymers. The hydrophilic sheath layer as well as the nanoscale size of a micelle is responsible for reducing the uptake by the reticuloendothelial system, prolonging the in vivo circulation duration and increasing specific accumulations within tumor tissues. The hydrophobic inner core of a micelle is beneficial to provide a high loading capacity of poorly water-soluble drugs and stabilize drugs that are sensitive to the surrounding environment [3]. However, recent studies have shown that self-assembled polymeric micelles lose their drug contents immediately after systemic administration, mainly due to the structural dissociation during blood circulation [4]. Many factors, such as high dilution, pH, temperature, ionic strength, large shear forces and the presence of

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numerous charged blood components, can affect their stability [5]. These interactions lead to an early disintegration or aggregation of micelles and cause a premature drug release before accessing cancer cells, which is the main reason for side effects and dramatic decreases in the therapeutic efficacy [6]. To reach the next level of clinical relevance, one of the most important ‘‘prerequisites” in the design of an efficient drug carrier is to stably retain the loaded drug in blood circulation before reaching the pathological site [7]. Covalent crosslinking between specific domains has been utilized to improve the structural stability of micelles, providing dramatic improvements in pharmacokinetics and biodistribution of encapsulated drugs [8]. In order to make the crosslinked micelles responsive to the microenvironment of tumor site or inside tumor cells, crosslinkers with intrinsic responsiveness to pH, temperature, or redox conditions have been used for micelle construction. The micelles exhibit an improved stability and drug retention in blood circulation, but the cleavage of intramicellar crosslinkers or disassembly of the micelles responding to the stimuli leads to an exclusive drug release in the target site [9]. Another strategy to alleviate the premature release from micelles is the chemical conjugation of hydrophobic anticancer drugs onto polymers, followed by self-assembling of the dugconjugated polymers into stable micelles. Drug-conjugate micelles (DCM) that combine the features of polymeric micelles and prodrugs could overcome the obvious problems of polymer–drug conjugates, such as the limited molecular mass, high molecular fluidity, and extended distribution in blood and fluids all over the body [10]. Camptothecin (CPT) and derivatives are known as topoisomerase-I inhibitors exhibiting high antitumor activities against a wide spectrum of human malignancies. One of the intrinsic limitations of CPT is the existence of a pH-dependent equilibrium between the lactone ring and an open carboxylate form, which shows less antitumor activity and several unpredictable side effects [11]. Various amphiphilic prodrugs of CPT with the propensity to self-assemble into micelles or vesicles have been exploited to address the issues [12]. Zhang et al. prepared azidefunctionalized CPT derivatives, followed by conjugation with poly (aspartic acid) derivatives containing alkyne groups by click cycloaddition to give DCM, indicating a low release due to the relatively stable linkers [13]. It is indicated there are significant differences in the glutathione (GSH) levels inside and outside tumor cells. GSH is produced intracellularly and maintained at mM levels in the cytosol and subcellular compartments, while the rapid enzymatic degradation limits GSH concentrations to lM in plasma [14]. To achieve a prompt release inside tumor cells, Li et al. prepared CPT conjugates with poly(ethylene glycol) (PEG) containing disulfide linkages between them. The self-assembled micelles showed a relatively high critical micelle concentration (CMC) of 60 lg/ml and a large initial burst release, due to a less optimal balance between the hydrophilic and hydrophobic segments [12]. To increase the targeting capabilities and stabilities of micelles, folic acid (FA)-decorated polymer–drug conjugates were synthesized with disulfide linkages between CPT and amphiphilic poly(ethylene glycol)-b-poly(e-caprolactone) (PECL) copolymers, showing a CMC of around 2 lg/ml. But the addition of hexadecanol during micelle formation was needed to modulate the interactions of hydrophobic segments in micelles and enhance the reductive sensitivity [15]. In our previous study, 4-armed PEG was used to construct multiarmed amphiphilic copolymers with drug conjugated, and the composition and arm structure showed significant effects on the micelle behaviors [16]. In view of CPT loading content, in vitro cellular uptake and reductive sensitivity, an optimal performance was obtained for micelles from heteroarm carriers with FA conjugation on 2 arms of PEG as targeting groups of micelles and copolymerization of e-caprolactone (e-CL) with PEG from the other 2 arms, followed by conjugation CPT through

disulfide linkages [16]. But a head-to-head comparison of the in vitro profiles and in vivo performance has never been launched for DCM and conventional micelles with drug loadings by physical encapsulation. In the current study, the carriers of drug-encapsulated micelles (DEM) were prepared from 4-armed PEG with FA conjugation on 2 arms of PEG as targeting groups of micelles. To achieve a structure similar to that of DCM carriers, the other 2 arms of PEG were conjugated with poly(e-caprolactone) (PCL) segments through disulfide linkages. The in vitro drug release, reductive sensitivity and cytotoxicity to tumor and normal cells of DCM were compared with those of DEM. The plasma drug concentrations were determined to reflect the bioavailability after intravenous injection of DCM and DEM in comparison with free drug administration. The tissue distributions, acute toxicity, antitumor and antimetastasis efficacies were investigated on tumor-bearing mice after administration of DCM and DEM. 2. Materials and methods 2.1. Materials and cells CPT was received from Knowshine Pharmachemicals Inc. (Shanghai, China), and GSH was from Aladdin (Beijing, China). Bovine serum albumin (BSA), human serum albumin (HSA), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide, trypsin, RNase, collagenase IV, and dialysis membrane (1 kDa cutoff) were procured from Sigma (St. Louis, MO). Rabbit anti-mouse antibodies of caspase-3 and Ki-67, goat antirabbit IgG–horseradish peroxidase (HRP) and 3,3-diaminobenzidine (DAB) developer were purchased from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Mouse breast carcinoma 4T1 cells and human umbilical vein endothelial cells (HUVECs) were obtained from American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 medium (Invitrogen, Grand Island, NY) supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco BRL, Grand Island, NY), 100 U/ml penicillin and 100 lg/ml streptomycin (Sigma, St. Louis, MO). All other chemicals and solvents were of reagent grade or better, and received from Changzheng Regents Co. (Chengdu, China), unless otherwise indicated. 2.2. Preparation of DCM and DEM DCM carriers were prepared from 4-arm-PEG with FA conjugation on two arms and PCL copolymerization on another two arms, followed by CPT conjugation through dithiodipropionic acid [16]. DEM carriers were prepared from 4-arm-PEG with FA conjugation on 2 arms and another 2 arms were conjugated with PCL through dithiodipropionic acid. The synthesis route, preparation process and characterization results of DEM carriers are provided in Supplementary information. DCM and DEM were fabricated by a solvent evaporation method as described previously with some modifications [16]. Briefly, 4 mg of DCM or DEM carriers was dissolved in 20 ml of tetrahydrofuran, and added dropwise into deionized water under vigorous stirring. The resulting suspension was kept under stirring overnight to remove tetrahydrofuran to form DCM or empty micelles of DEM carriers. DEM were obtained by the similar method as above through dissolving 0.4 mg of CPT and 4 mg of DEM carriers into tetrahydrofuran. The unencapsulated CPT was removed by dialysis to obtain DEM. 2.3. Characterizations of micelles The average size and size distribution of micelles were performed by dynamic light scattering (DLS, Nano-ZS90, Malvern Ltd., UK). The morphologies of micelles were observed by

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transmission electron microscopy (TEM, JEM-2100F, JEOL, Tokyo, Japan) operated at 200 kV. The samples were prepared by dropping micelle suspensions on a copper grid followed by staining with phosphotungstic acid. Micelles were lyophilized and then dissolved in dimethyl sulfoxide (DMSO), and the fluorescent intensities were measured by a fluorospectrophotometer (Hitachi F-7000, Japan), at the excitation wavelength of 360 nm and the emission wavelength of 450 nm. The CPT loading content in micelles was obtained after comparing with fluorescent intensities of CPT solutions with series known concentrations [17]. The CMCs of DCM and DEM carriers were determined by fluorescence spectroscopy using pyrene as a fluorescence probe as described previously [16]. 2.4. In vitro drug release from micelles The CPT release from micelles was performed in phosphate buffer saline (PBS) in the presence of GSH and/or HSA. Briefly, 1 ml of DCM and DEM was transferred into dialysis bags, which were immersed in 30 ml of media to acquire a sink condition. The release testing was performed in PBS, PBS containing 10 mM of GSH (PBS/GSH), PBS containing 4% HSA (PBS/HSA), and PBS containing both 10 mM of GSH and 4% HSA (PBS/GSH/HSA), which were maintained in a shaking water bath at 37 °C. At predetermined time intervals, 1 ml of the release media was withdrawn and 1 ml of fresh media was added for continuous incubation. The amount of CPT release into the media was determined by a fluorospectrophotometer as above. 2.5. Stability of micelles and drugs released from micelles The stability of micelles was investigated from the size changes in the presence of 4% HSA [18]. DCM and DEM were incubated in PBS or PBS/HSA at 37 °C. At predetermined time intervals, micelle suspensions were withdrawn for DLS analysis as above. The structural integrity of released drugs was detected in the presence of HSA by circular dichroism (CD) as described previously [19]. Briefly, to obtain the release drug from DCM, micelles were incubated with PBS containing 40 mM GSH at 37 °C for 72 h to break the disulfide linkages completely, followed by extraction with DMSO. Then, 2 lg/ml of the release drugs was mixed with 4% HSA and incubated for 24 h, followed by determination with a CD spectrometer (Jasco J-810, Japan). The spectra were scanned between 300 and 450 nm with an optical path length of 0.1 cm and sensitivity of 2 m°/cm. 2.6. In vitro cellular uptake of micelles by 4T1 cells In vitro cellular uptake was performed on 4T1 cells by using flow cytometry as described previously [20]. Briefly, 4T1 cells were seeded in 6-well tissue culture plates (TCPs) at 1  106 cells/well and allowed to grow for 24 h. DCM and DEM with a final CPT concentration of 10.0 lg/ml were added to each well and incubated for 4 h, using free CPT as the control. Cells were then washed with cold PBS twice and harvested by 0.25% (w/v) trypsin. After collection by centrifugation at 4 °C, cells were suspended in PBS containing 2% FBS. The cell samples were quickly analyzed by flow cytometry (FACSCalibur, BD Biosciences, CA). Data were collected of 5000 gated events and analyzed with the Flowjo 7.0 software. 2.7. In vitro cytotoxicity of micelles and drugs released from micelles The in vitro cytotoxicity of DCM and DEM was investigated on 4T1 and HUVECs, using free CPT and empty micelles as the control. Briefly, cells were seeded in a 96-well TCP at a density of 5000 cells/well, and incubated for 24 h. The media were removed

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and replenished by media containing free CPT, DCM or DEM with final CPT concentrations from 10 ng/ml to 10 lg/ml. Cells were also treated by empty micelles with the same concentrations to DEM. After culture for 48 h and PBS wash, cells were incubated with 100 lL of media containing MTT in each well for 4 h at 37 °C. Then, 150 lL of DMSO was added to dissolve the formed crystals and the absorbance intensity at 570 nm was recorded by a microplate spectrophotometer (Elx-800, Bio-Tek Instrument Inc., Winooski, VT). The in vitro cytotoxicity of the released drug from DCM was also measured from cell availability and cell cycle analysis and compared with that of free CPT. Briefly, the released drug from DCM was prepared as above and adjusted to 10 lg/ml by culture media. The cell availability was detected by MTT assay as above. For the cell cycle analysis, cells were seeded in a 6-well TCP at 1  106 cells/well and allowed to grow for 24 h. The culture media were replenished with media containing 10 lg/ml of the released drug or free CPT, using untreated cells as the control. After incubation for 48 h, cells were harvested by trypsinization, fixed in ice-cold 70% ethanol, and deposited for 12 h at 4 °C to increase the penetrability of cell membrane. Then, cells were harvested by centrifugation and washed with PBS before being resuspended in 0.5 ml of PBS containing 50 lg/ml of propidium iodide and 100 mg/ml of RNase. After incubation at 37 °C for 30 min, cell samples were detected by a flow cytometer (Accuri C6, BD Biosciences, CA). 2.8. Pharmacokinetic analysis of micelles after intravenous injection Pharmacokinetic profiles of micelles were determined after intravenous injection as described previously [21]. Female Sprague-Dawley rats weighing 180–220 g were from Sichuan Dashuo Biotech Inc. (Chengdu, China), and all animal protocols were approved by the University Animal Care and Use Committee. Briefly, DCM and DEM were injected through tail veins at a single dose of 4.0 mg CPT/kg body weight, using free CTP as the control. After injection for 0.25, 1, 4, 8, 12, 24, 48, and 72 h, blood samples of around 1 ml were collected in heparinized tubes and centrifuged to recover plasma, and the CPT concentration was measured as described previously [22]. Important parameters including the maximum concentration (Cmax), half-life (T1/2b), clearance (CL), area under the curve (AUC0–1), and mean resident time (MRT) were calculated by the PK solver software using a bicompartmental model as described previously [23]. 2.9. Tissue distributions of CPT after micelle treatment The tissue distribution, acute toxicity, tumor treatment and metastasis inhibition were determined on tumor-bearing mice after intravenous injection of micelles. Female Balb/c mice weighing 18–22 g were from Sichuan Dashuo Biotech Inc. (Chengdu, China). 4T1 cells were expanded in RPMI 1640 media containing 10% FBS, and the collected cells were suspended in PBS at 1  107 cells/ml. Xenograft 4T1 tumors were established by injection of 100 lL of cell suspensions into the mammary fat pad of each mouse, and the distribution of CPT in tumor and other tissues at different time points was determined as described previously with some modifications [24]. Briefly, after the tumor growth reached around 100 mm3 in volume, CPT, DCM or DEM were injected into tumor-bearing mice by tail veins at a dose of 4 mg CPT/kg. After injection for 4 and 24 h, animals were sacrificed to retrieve heart, liver, spleen, lung, kidney, and tumor tissues. Tissues were cut into small pieces, washed with ice-cold saline to remove bloodstains, and weighed. The tissue pieces were homogenized with saline and acidified to pH 3.0 with acetic acid, then DMSO was added in the tissue homogenate for continued homogenization. The above mixtures were vortexed for 3 min and centrifuged for 5 min at 3000 r/min. The amount of CPT in the supernatant and plasma

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was measured by a fluorospectrophotometer as above. The amount of CPT in a tissue was obtained using a standard curve from known concentrations of CPT in the homogenate of this tissue from untreated mice. The percentage of injected dose (ID%) indicated the ratio of actual amount of drug in a tissue to the total amount of injected drug. 2.10. In vivo antitumor efficacy after micelle treatment 4T1 tumors were established as above, and animals were randomly divided into 5 groups with 7 mice per group. CPT, DCM or DEM were injected through tail veins at a dose of 4 mg CPT /kg on days 0, 2, 4, and 6, using empty micelles and saline injection as the control. The body weights, tumor volumes and survival rates of animals were monitored every other day after treatment. The lengths of the major axis (longest diameter) and minor axis (perpendicular to the major axis) of a tumor were measured with a vernier caliper, and the tumor volume was calculated as described previously [25]. The number of live animals at each time point was plotted in Kaplan–Meier survival curves, and the 50% mean survival time was obtained for a comparison of the treatment efficacy. On day 21 after treatment, animals were sacrificed to retrieve tumors and lungs, which were fixed by 10% formaldehyde solution and processed routinely into paraffin blocks. Tissue sections with a thickness of 4 lm were stained with hematoxylin and eosin (HE) and observed with a light microscope (Nikon Eclipse E400, Japan). To investigate the cell proliferation and apoptosis in tumors, immunohistochemical (IHC) assessment of Ki-67 and caspase-3 was conducted on tumor sections as described previously [25]. The slides were counterstained for 1 min with hematoxylin and then dehydrated with sequential ethanol for sealing and microscope observation. To quantify the protein expressions, Ki-67 or caspase-3-positive cells were counted in five randomly selected areas from IHC staining images, and compared with the total number of cells in these areas. 2.11. In vivo toxicity of micelle treatment The in vivo toxicity of micelle treatment was determined on tumor-bearing mice with respect to the hematological analysis and histological analysis of small intestine. Briefly, CPT, DCM or DEM were injected into the established tumor models as above. At 24 h after the last injection, around 1.0 ml of blood was collected for the determination of hematological parameters, including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT). Blood was centrifuged at 3000 rpm at 4 °C to collect serum for biochemical analysis of aspartate transaminase (AST), alanine transaminase (ALT), urea nitrogen (BUN), and creatinine (CRE) as described previously [26]. Additionally, acute or chronic diarrhea could be induced by CPT [27]; thereby, small intestines were also retrieved for morphological observation after HE staining as indicated above. 2.12. In vivo antimetastasis efficacy after micelle treatment The numbers of metastatic 4T1 cells in lungs were determined by a clonogenic assay as described previously [28]. Briefly, lungs were retrieved after treatment for 21 days and digested in 5 ml of PBS containing 1 mg/ml of collagenase IV for 2 h at 37 °C on a platform rocker. After filtration through nylon cell-strainers with a mesh size of 70 lm, the resulting cells were suspended and plated with a series of dilutions in RPMI 1640 media containing 60 mM thioguanine (Meilun Biotechnology Ltd., Dalian, China) for clonogenic growth. Ten days later, the clonogenic cells were fixed

with methanol and stained with 0.03% methylene blue for counting. 2.13. Statistical analysis The results are reported as mean ± standard deviation (SD). Whenever appropriate, comparisons among multiple groups were performed by analysis of variance (ANOVA), while a two-tailed Student’s t-test was used to discern the statistical difference between two groups. A probability value (p) of less than 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Characterization of DCM and DEM Fig. 1a illustrates the structure of DCM and DEM carriers, and the schematic drawing of the micelle formation is shown in Fig. 1b. Amphiphilic DCM carriers tended to self-assemble into micelles with hydrophobic cores of CPT and PCL segments and hydrophilic PEG shells in aqueous solution. DEM carriers with hydrophobic PCL and hydrophilic PEG were self-assembled into micelles in water, encapsulating CPT in the hydrophobic cores. The CPT loading content in DEM was about 7.8 ± 0.4% and close to that of DCM at around 7.5%, estimated from 1H NMR spectra [16]. The CMC values of DCM and DEM were 0.93 and 0.97 lg/ml, respectively. TEM observations indicated an uniform and spherical shape of selfassembled micelles with an average size of 30–40 nm (Fig. 1c). As shown in Fig. 1d, the average diameters of DCM and DEM were about 130 nm determined by DLS in aqueous suspensions, and the smaller size observed from TEM images was due to the volume shrinkage of micelles after vacuum-drying. 3.2. In vitro drug release profile and stability of micelles In this study, the release profile of CPT from DCM and DEM was examined in PBS in the presence of 10 mM GSH and/or 4% HSA. As shown in Fig. 2a, DCM and DEM showed similar drug release profiles after incubation in PBS except a slightly higher release from DEM during the initial period. During 3 h of incubation in PBS, around 15% and 25% of drug release were detected from DCM and DEM, respectively. Both of the micelles showed reductionsensitive drug release, and the release enhancement was more significant for DEM after incubation with 10 mM GSH. There was around 43.5% of CPT release from DEM during 2 h, while DCM showed an accumulated release of around 41.8% during 5 h of incubation with GSH. In addition, compared to over 80% of CPT release from DEM during 12 h, a gradual drug release from DCM was indicated during 120 h of incubation. The lower burst release and less significant reduction sensitivity of DCM were due to the conjugation of CPT with PCL segments by disulfide linkages, which were embedded in the hydrophobic cores of micelles [15]. The lactone ring of CPT is prone to hydrolysis under physiological condition, and a pH-dependent equilibrium exists with an open carboxylate form [11]. HSA is shown to bind preferentially with the carboxylate form with a 150-fold higher affinity than the lactone form of CPT in PBS at 7.4 [29]. Thus, the release of CPT from DCM and DEM was investigated in the presence of HSA within dialysis bags (1 kDa cutoff) to reflect the structural integrity of CPT. As shown in Fig. 2a, there was a slower release of CPT from DCM and DEM after incubating HSA into the release media, and the release retardant was more significant for DEM. The CPT derivative released from DCM was CPT-SH after disulfide breakdown, and the acylation of 20-OH group of CPT might block the shift of equilibrium in favor of the carboxylate form, leading to fewer bindings

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Fig. 1. (a) The molecular structures of DCM and DEM carriers. (b) Schematic drawing of the formation of DCM and DEM. (c) Typical TEM image of DCM. (d) Size distributions of DCM and DEM measured through dynamic light scattering.

with HSA [30]. Compared with around 95% of CPT release from DEM during incubation with 10 mM GSH for 48 h, the addition of HSA into the release media led to a significantly lower release of around 45.8% (p < 0.05). Thus, although micelles may protect the activity of CPT lactone to some extent [31], higher amount of carboxylate form was shown for CPT released from DEM than that of DCM. The lactone structure of the released drugs from DCM and DEM was also detected by CD approach in the presence of HSA. As shown in Fig. 2b, the interaction of carboxylate CPT led to a longwavelength shift from 394 to 380 nm after interaction with HSA. There were no significant changes in the absorption band at around 380 nm for those released from DCM in the presence of HSA, indicating the retention of lactone rings in CPT-SH [19]. In addition, the band at around 340 nm became smaller and totally disappearing on progressing from the lactone to the carboxylate form [19], while CPT-SH indicated an absorption peak at around 340 nm in the presence of HSA. Thus, the CD spectra indicated that the CPT-SH released from DCM may stabilize the lactone structure and protect from hydrolysis. For uses in the drug solubilization and target delivery, micelles should remain intact during formulation and administration. The size stability of DCM and DEM was investigated in the current study by DLS. As shown in Fig. 2c, there were no significant changes in the size of DCM during incubation with HSA for 72 h. However, the size of DEM decreased from 130 to 95 nm after

incubation for 72 h in the presence of HSA. The size decrease can be attributed to the disintegrity of DEM and the release of CPT from micelles [32]. 3.3. In vitro cellular uptake and cytotoxicity of micelles Cellular uptake of DCM and DEM was performed on 4T1 cells by flow cytometry. As CPT is auto-fluorescent, it can be used directly to measure the cellular uptake without introducing additional fluorescent probes. Fig. 3a shows the histogram of fluorescence measured on 4T1 cells after incubation with either free CPT, DEM or DCM at an equivalent CPT concentration of 10 lg/ml for 4 h, using cells without any treatment as the control. 4T1 cells after incubation with both of the micelles showed higher fluorescence intensities than those with free CPT due to the targeted recognition of folate receptors on cells [16]. A slightly stronger fluorescence was detected for DEM, which could be attributed to the slightly higher loading content of CPT in DEM than that in DCM. In vitro cytotoxicity of DCM and DEM was conducted on 4T1 and HUVECs as a tumor and normal cell model, respectively. Fig. 3b summarizes the cell viabilities against various concentrations of equivalent CPT ranging from 0.1 to 10 lg/ml, using empty micelles as the control. The IC50 values of 4T1 cells after treatment for 48 h with DCM, DEM, and free drug were 1.86, 2.85, and 2.98 lg/ml, respectively. The lower IC50 values of free CPT should be due to the fact that CPT was available only after the breakdown of DCM

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Fig. 2. (a) The percent release of CPT from DCM and DEM in PBS and in the presence of 10 mM GSH and/or 4% HSA (n = 3). (b) Circular dichroism spectra of released drugs from DCM and DEM in PBS and in the presence of 4% HSA. (c) Typical DLS images of DCM and DEM obtained before and after incubation at 37 °C in PBS and in the presence of 4% HSA.

and DEM [16]. The IC50 values of HUVECs were 6.53, 3.15 and 0.97 lg/ml after treatment with DCM, DEM, and free drug, respectively. Few FA receptors were found to be located on the membrane of HUVECs than those on 4T1 cells [33], leading to no apparent enhancement in the cellular uptake of micelles. Thus, DCM showed significantly higher cytotoxicities to 4T1 cells and significantly lower cytotoxicities to HUVECs than free drug (Fig. 3b). In addition, due to the lower GSH levels in HUVECs than 4T1 cells [6], the slower breakage of disulfide linkages led to a lower drug release from DCM than that from DEM, resulting in a lower cytotoxicity of DCM to HUVECs. 3.4. In vitro cytotoxicity of drugs released from DCM The breakdown of disulfide linkages in DCM leads to the release of CPT-SH, and the b-thioester bonds between b-thiopropionic acid and CPT are easily hydrolyzed to release the active moiety CPT by esterase, which is abundant in cells [34]. In order to demonstrate the antitumor ability of the released drug from DCM, the cytotoxicity and cell cycle assay were conducted on 4T1 cells and compared with those released from DEM. Fig. 4a shows the cell viabilities after treatment with the released drugs of different concentrations. The IC50 values of the released drugs from DCM and DEM were 2.85 and 3.02 lg/ml, respectively, indicating that CPT-SH had a similar cytotoxicity to CPT. As shown in Fig. 4b, around 31.8% and 33.1% of 4T1 cells were arrested in G2/M, and 7.3% and 9.6% of apoptotic cells were induced by CPT-SH and CPT, respectively. It was indicated that the released drugs from DCM showed cytotoxicities and cytotoxic mechanisms similar to CPT on tumor cells. 3.5. Pharmacokinetic evaluation of micelles Fig. 5a shows the plasma concentration–time profiles of DCM, DEM and free CPT after intravenous injection, which showed biphasic curves with a rapid distribution phase, followed by a slow elimination phase. The injection of free CPT gave plasma

concentrations of 28.2 lg/ml after 15 min, which was slightly higher than that after treatments with DCM and DEM, at plasma concentrations of 25.9 and 27.9 lg/ml, respectively. There was more significant decrease in the plasma concentrations after injection of free CPT, and the CPT concentrations were 2.64, 3.77, and 7.17 lg/ml after administration of free CPT, DEM, and DCM for 12 h, respectively. The CPT content in plasma was undetectable after CPT injection for 48 h, but slight decreases in the plasma concentrations of CPT were detected for DCM and DEM, at 2.87 and 0.34 lg/ml after 72 h, respectively. Table 1 summarizes the pharmacokinetic parameters including T1/2b, AUC0–1, MRT and CL after administration of free CPT, DCM and DEM. Both of CPT-loaded micelles showed significantly higher T1/2b, AUC0–1, and MRT, and significantly lower CL values than those of free CPT (p < 0.05). The encapsulation of CPT in micelles resulted in a significant prolongation of CPT presence in plasma [35]. In addition, the T1/2b, AUC0–1 and MRT values after DCM treatment were around 1.7, 2.65, and 2.07 folds higher than those from DEM (p < 0.05), respectively. DCM treatment led to around 2.66 folds lower clearance than DEM (p < 0. 05). These results showed that DCM had a significantly longer blood circulation time than DEM. Fig. 5b summarizes the tissue distributions of CPT-loaded micelles in tumor-bearing mice. After a single dose of CPT-loaded micelles for 4 h, the liver, spleen and lung showed significantly higher CPT concentrations than those after CPT injection (p < 0.05). This was due to the removal of micelles by macrophages in these tissues [36]. The CPT levels in kidney after injection of free CPT were significantly higher than those after micelle treatment for 4 h (p < 0.05), but there was no significant difference in the CPT accumulation between heart and tumor (p > 0.05). After 24 h of injection, there were rapid decreases of CPT concentrations in heart, liver, spleen, lung and kidney. Though there were significantly higher CPT levels in liver, spleen and lung after micelle treatment than those after free CPT injection (p < 0.05), there were no significant differences between DCM and DEM (p > 0.05). Compared with those after 4 h, the accumulation of CPT in tumors

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derivatives can induce severe inhibition of marrow leading to the change of hematological parameters [37]. As shown in Fig. 6a, free CPT caused a significant decrease in WBC and HGB compared to the control (p < 0.05), but only a slight decrease was indicated for CPTloaded micelles, especially for DCM. ALT and AST are important enzymes in liver and usually used to monitor chronic liver diseases [26]. An increase of serum BUN and creatinine concentrations is the marker for kidney damage and nephrotoxicity [38]. As shown in Fig. 6a, the levels of AST, ALT, BUN and creatinine showed a slight increase after micelle treatment, but a significant increase after CPT administration compared to those of normal mice. In addition, the levels of these biochemical parameters were slightly higher after DEM treatment than those of DCM, which may be the different release profiles (Fig. 2) and distributions in liver and kidney (Fig. 5). The change in body weights as a function of time in tumorbearing mice was used as one of the markers of safety [39]. As shown in Fig. 6b, the body weights of animals experienced a dramatic loss after treatment with free CPT, and even remained declining on the days of cessation of treatment. However, there was a slight weight loss in the groups received micelles, and the body weights were gradually recovered after cessation of treatment, indicating a slight systemic toxicity after DCM and DEM administration. It was indicated that acute diarrhea can be induced by CPT and its derivatives [27]. On day 7 after intravenous injection, mice were sacrificed and parts of the small intestine were sampled for pathologic studies. As shown in Fig. 6c, the intestinal morphology and integrity were severely disrupted after CPT treatment, with complete abolishment of the top villi along with severe inflammation. The small intestinal mucosa of mice after DEM

Fig. 3. (a) Typical histogram of fluorescence intensities measured on 4T1 cells after incubation with free CPT, DCM and DEM. (b) The cytotoxicity of free CPT, DCM, DEM and empty micelles (EM) to 4T1 and HUVECs (n = 5).

increased after micelle treatment for 24 h. The CPT concentration in tumors after DCM treatment reached 3.67 lg/g tissue, which was 1.87 and 7.97 folds higher than those after injection of DEM and free CPT, respectively (p < 0.05), which may be due to the prolonged blood circulation of DCM and reduced initial drug release from micelles. 3.6. In vivo toxicity of micelle treatment For acute toxicity study, different formulations of CPT were intravenously injected into tumor-bearing mice with equivalent concentration of CPT at 4.0 mg/kg. It is reported that CPT and its

Fig. 4. (a) The cytotoxicity of drugs released from DCM and DEM to 4T1 cells (n = 5). (b) The cell cycle distribution and the apoptotic rate of 4T1 cells after treatment by drugs released from DCM and DEM for 48 h, compared with cells without treatment.

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3.7. Antitumor efficacy of micelle treatment

Fig. 5. (a) Pharmacokinetics of free CPT, DCM and DEM after intravenous administration to rats (n = 5). (b) The distribution of CPT in heart, liver, spleen, lung, kidney and tumors at 4 and 24 h after intravenous administration of free CPT, DCM and DEM to 4T1 tumor-bearing mice at a dose of 4.0 mg CPT/kg body weight (n = 4). ⁄p < 0.05.

Table 1 Pharmacokinetic parameters of CPT after intravenous administration to rats of free CPT, DCM and DEM (n = 5).

DCM DEM CPT * # $

T1/2b (h)

AUC0–1 (h lg/ml)

MRT (h)

CL (ml/h)

Cmax (lg/ml)

28.6 ± 1.3*,# 15.7 ± 2.4$ 9.2 ± 0.7

524.2 ± 37.1*,# 197.2 ± 16.4$ 124.3 ± 10.5

38.3 ± 3.6*,# 18.5 ± 2.1$ 9.1 ± 0.6

3.8 ± 0.2*,# 10.1 ± 0.8$ 16.1 ± 2.3

30.2 ± 7.1 24.7 ± 2.5 25.6 ± 3.6

p < 0.05 (DCM vs DEM). p < 0.05 (DCM vs CPT). p < 0.05 (DEM vs CPT).

treatment showed fibrotic changes, deformed glandular alignment, and glandular duct disappearance. On the other hand, the small intestinal mucosa of DCM-treated mice showed only mild shortening, mild inflammatory cell invasion, and a slight decrease in the villi numbers compared with the normal tissues.

The antitumor efficacy of micelles was evaluated with respect to the inhibition of tumor growth, the survival rate of animals, and the histological and IHC analysis of tumors retrieved. Fig. 7a summarizes the tumor growth curves after intravenous injection of DCM, DEM and free CPT, using empty micelles and saline as the control. There were no significant differences in the tumor volumes of mice treated with empty micelles and saline (p > 0.05), reaching around 2000 mm3 after 27 days. The tumor growth was significantly inhibited after treatment with DCM and DEM during the observation period compared with that of CPT (p < 0.05). The improved antitumor activity should be attributed to the active targeting achieved by FA grafts on the micelles. In addition, DCM showed superior antitumor efficacy than DEM, and the tumor volume reached about 780 and 1050 mm3 after treatment with DCM and DEM for 30 days, respectively (p < 0.05). Fig. 7b summarizes the survival rate presented in a Kaplan–Meier plotting, indicating that the micelle treatment could prolong the life of tumor-bearing mice compared with the control. The 50% mean survival time of animals treated with PBS and empty micelles was 20 and 23 days, respectively. The CPT treatment led to no apparent changes in the animal survival, and 50% of the mice died within 23 days, in line with the toxicity of free CPT. However, the treatment with DCM and DEM showed 50% mean survivals for 43 and 39 days, respectively. In order to further indicate the antitumor efficacy, tumors were retrieved after 21 days of treatment for HE and IHC analyses. As shown in Fig. 7c, there were large amount of living cells (blue area)1 in tumors after treatment with PBS and empty micelles. Apparent necrotic areas were shown in tumors after treatment with free CPT, DEM and DCM. Necrosis within tumors represents a significant prognostic factor of tumor shrinkage after chemotherapy [40]. The necrotic region after DCM treatment was larger than other groups, along with apparent vacuolus degeneration of tumor cells. The CPT-induced apoptosis is thought to be generally dependent on the release of cytochrome c and the subsequent activation of caspase-3 [41]. Fig. 7d shows the IHC staining of caspase-3 on tumors after treatment for 21 days, and the positively stained cells were counted. The treatment with empty micelles and saline led to around 11.3% and 7.4% of apoptotic cells, respectively. Compared with around 47.9% of apoptotic cells in tumors after free CPT treatment, significantly higher amount of apoptotic cells was determined after treatment with DEM and DCM, at around 68.3% and 83.5% of apoptotic cells, respectively (p < 0.05). Ki-67 is an antigen that corresponds to a nuclear non-histone protein expressed by cells in the proliferative phases, and a higher index of Ki-67 means a faster proliferation [42]. Fig. 7e shows typical IHC staining images of Ki-67 in tumor tissues after 21 days of treatment. As expected, significantly higher numbers of Ki-67-stained cells were observed in tumors treated with empty micelles and saline, at around 90.5% and 85.6% of Ki67-stained cells, respectively, compared with those after free drug treatment (p < 0.05). A significantly lower proliferation index was determined for DCM-treated tumors at around 13.6%, compared with 51.5% and 27.9% of Ki-67-positive cells for tumors after treatment with free CPT and DEM, respectively (p < 0.05). 3.8. Antimetastatic efficacy of micelle treatment 4T1 is highly invasive breast cancer cell lines and tends to metastasize and colonize in distant organs including the lung, bone, brain, and liver, primarily through a hematogenous route [28]. Fig. 8a shows HE staining results on lung sections after 1 For interpretation of color in Fig. 7, the reader is referred to the web version of this article.

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Fig. 6. (a) The hematological and biochemical parameters of blood retrieved on day 7 after intravenous administration of free CPT, DCM and DEM to 4T1 tumor-bearing mice at a dose of 4.0 mg CPT/kg body weight, using empty micelle (EM) and saline injection as the control, compared with normal mice (N.M.; n = 4). (b) Body weight changes of tumor-bearing mice after intravenous injection of free CPT, DCM and DEM, using EM and saline injection as the control (n = 6). (c) Typical HE staining images of small intestines retrieved from tumor-bearing mice on day 7 after intravenous injection of free CPT, DCM and DEM, using saline injection as the control. Arrows indicate the shortened intestinal villi, and scale bar represents 20 lm.

Fig. 7. (a) Tumor growth and (b) survival curves of 4T1 tumor-bearing mice after intravenous administration of free CPT, DCM and DEM at a dose of 4.0 mg CPT/kg body weight, using empty micelle (EM) and saline injection as control. (c) Typical HE staining images (‘‘N” represents necrotic area, ‘‘T” represents tumor mass), (d) IHC staining images of caspase-3 and (e) Ki-67 of tumors retrieved on day 21 after intravenous administration of free CPT, DCM and DEM, using EM and saline injection as the control. Bars represent 20 lm.

treatment for 21 days. Apparent metastases of 4T1 cells were found in the lung sections of mice after treatment with empty micelles and saline. The lung metastasis was less significant in

mice after treatment with CPT and CPT-loaded micelles, and DCM indicated the least metastasis in lung sections with normal alveolar spaces. The number of metastatic cells in the lungs could

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[43]. In the current study, DCM micelles were proposed to overcome the premature drug release and enhance the accumulation in tumor tissues. Both DCM and DEM carriers showed a similar star structure, and 2 arms of 4-arm PEG were conjugated with FA as targeting groups of micelles. To construct DCM carriers, the other 2 arms of PEG were copolymerized with PCL, followed by conjugation CPT through disulfide linkages [16]. Alternatively, the other 2 arms of PEG were conjugated with PCL segments through disulfide linkages to obtain DEM carriers (Scheme S1). It should be noted that the stoichiometric ratio of the heteroarm structure should be prone to statistics. Both DCM and DEM had similar sizes, drug loadings and CMC values, and similar cellular uptake efficiencies were detected due to the FA-mediated endocytosis (Fig. 3a). To achieve a deep understanding of DCM, comprehensive comparisons with DEM were initially launched with respect to the in vitro and in vivo performances. Firstly, there was larger initial release from DEM after incubation in PBS (Fig. 2a), and the presence of HSA led to a lower stability of DEM during in vitro incubation (Fig. 2c). After intravenous administration, larger T1/2b and AUC0–1 and a lower clearance were determined for DCM than those of DEM (Table 1). The prolonged blood circulation led to a higher drug accumulation in tumor tissues for DCM than that of DEM (Fig. 5b). Secondly, compared with DCM, DEM indicated higher sensitivity of drug release to reductive environment (Fig. 2a), resulting in more significant toxicities to normal cells (Fig. 3b). Thus, although DCM and DEM showed similar distribution profiles in normal tissues (Fig. 5b), DEM indicated higher toxicities to liver, kidney and small intestine (Fig. 6). Thirdly, compared with CPT, the release drugs from DCM showed similar cytotoxic profiles to 4T1 cells (Fig. 4), due to the hydrolysis of b-thioester bonds between b-thiopropionic acid and CPT by esterase inside cells to release CPT [33]. In addition, the acylation of 20-OH group in CPT-SH benefited to remain the lactone ring of CPT in the presence of HSA (Fig. 2b). Thus, the increased tumor accumulation of DCM (Fig. 5b) and the enhanced structural integrity of release drugs from DCM (Fig. 2b) led to significantly higher antitumor efficacy (Fig. 7) and antimetastasis activity (Fig. 8) after DCM treatment than those of DEM. 4. Conclusions

Fig. 8. (a) Typical HE staining images of lung sections (‘‘T” represents metastatic tumors, ‘‘A” represents alveolar spaces, and ‘‘normal” represents lung tissue from a normal mouse; Bars represent 20 lm) and (b) the number of metastatic colonies in lungs retrieved on day 21 from 4T1 tumor-bearing mice after intravenous administration of free CPT, DCM and DEM, using empty micelle (EM) and saline injection as the control. ⁄p < 0.05.

be enumerated because 4T1 cells are resistant to 6-thioguanine, whereas normal cells are not resistant and die [28]. As shown in Fig. 8b, compared with empty micelle and saline treatment, significantly lower numbers of metastatic 4T1 cells were detected in lungs of mice after treatment with CPT and CPT-loaded micelles (p < 0.05). DCM indicated significantly lower numbers of colonies at around 940 than those from CPT and DEM-treated mice, at around 3250 and 2240, respectively (p < 0.05).

The in vitro and in vivo performances after DCM treatment were determined in comparison with DEM. Compared with DCM, DEM showed larger initial drug release and higher sensitivities of drug release to GSH and HSA inoculations. The release drugs from DCM indicated a cytotoxicity profile similar to CPT and remained a higher stability of lactone ring than CPT in the presence of HSA. DCM showed slightly higher cytotoxicities to 4T1 cells and significantly lower cytotoxicities to HUVECs than DEM. Compared with DEM after intravenous administration, DCM showed a prolonged blood circulation, a higher accumulation in tumor tissues, and a lower toxicity to normal tissues, and achieved a better antitumor efficacy and antimetastasis activity. Therefore, DCM provided a strategy to overcome the limitation of conventional DEM to some extent by alleviating the premature drug release during blood circulation, relieving the systemic toxicity and promoting the therapeutic efficacy.

3.9. Differences in treatment efficacy and systemic toxicity between DCM and DEM

Acknowledgments

The use of polymer–drug conjugates is effective to alleviate the premature release of therapeutics before reaching the disease sites. Patri et al. demonstrated that covalently conjugated drug to dendrimer was better suited for specifically targeted drug delivery in comparison with a dendrimer/drug inclusion complex in vitro

This work was supported by National Natural Science Foundation of China (21274117 and 31470922), Specialized Research Fund for the Doctoral Program of Higher Education (20120184110004), and Fundamental Research Funds for the Central Universities (2062015YXZT06).

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