Investigating the mechanism of enhanced cytotoxicity of HPMA copolymer–Dox–AGM in breast cancer cells

Journal of Controlled Release 117 (2007) 28 – 39 www.elsevier.com/locate/jconrel

Investigating the mechanism of enhanced cytotoxicity of HPMA copolymer–Dox–AGM in breast cancer cells Francesca Greco a,b,1 , María J. Vicent a,2 , Siobhan Gee a , Arwyn T. Jones a , Julia Gee b , Robert I. Nicholson b , Ruth Duncan a,⁎ b

a Centre for Polymer Therapeutics, Welsh School of Pharmacy, Redwood Building, Cardiff University, King Edward VII Ave., Cardiff CF10 3XF, UK Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Redwood Building, Cardiff University, King Edward VII Ave., Cardiff CF10 3XF, UK

Received 12 July 2006; accepted 5 October 2006 Available online 14 October 2006

Abstract Recently we have described an HPMA copolymer conjugate carrying both the aromatase inhibitor aminoglutethimide (AGM) and doxorubicin (Dox) as combination therapy. This showed markedly enhanced in vitro cytotoxicity compared to the HPMA copolymer–Dox (FCE28068), a conjugate that demonstrated activity in chemotherapy refractory breast cancer patients during early clinical trials. To better understand the superior activity of HPMA copolymer–Dox–AGM, here experiments were undertaken using MCF-7 and MCF-7ca (aromatase-transfected) breast cancer cell lines to: further probe the synergistic cytotoxic effects of AGM and Dox in free and conjugated form; to compare the endocytic properties of HPMA copolymer–Dox–AGM and HPMA copolymer–Dox (binding, rate and mechanism of cellular uptake); the rate of drug liberation by lysosomal thiol-dependant proteases (i.e. conjugate activation), and also, using immunocytochemistry, to compare their molecular mechanism of action. It was clearly shown that attachment of both drugs to the same polymer backbone was a requirement for enhanced cytotoxicity. FACS studies indicated both conjugates have a similar pattern of cell binding and endocytic uptake (at least partially via a cholesterol-dependent pathway), however, the pattern of enzyme-mediated drug liberation was distinctly different. Dox release from PK1 was linear with time, whereas the release of both Dox and AGM from HPMA copolymer–Dox–AGM was not, and the initial rate of AGM release was much faster than that seen for the anthracycline. Immunocytochemistry showed that both conjugates decreased the expression of ki67. However, this effect was more marked for HPMA copolymer–Dox–AGM and, moreover, only this conjugate decreased the expression of the anti-apoptotic protein bcl-2. In conclusion, the superior in vitro activity of HPMA copolymer–Dox–AGM cannot be attributed to differences in endocytic uptake, and it seems likely that the synergistic effect of Dox and AGM is due to the kinetics of intracellular drug liberation which leads to enhanced activity. © 2006 Elsevier B.V. All rights reserved. Keywords: Polymer–drug conjugates; Polymer therapeutics; Tumour targeting; Endocrine therapy; Chemotherapy; Breast cancer

1. Introduction A growing number of polymer–drug conjugates have entered clinical trial as antitumour agents (reviewed in [1–4]) and a poly (glutamic acid)–paclitaxel conjugate (XYOTAX™) is currently showing particular promise for the treatment of non small cell lung cancer (NSCLC) in women [5]. Due to their changed ⁎ Corresponding author. Tel.: +44 2920874180; fax: +44 2920874536. E-mail address: [email protected] (R. Duncan). 1 Current address: School of Pharmacy, University of Reading, Whiteknights, PO Box 226, Reading, Berkshire, RG6 6AP, UK. 2 Current address: Centro de Investigación Príncipe Felipe, Polymer Therapeutics Laboratory, Medicinal Chemistry Unit, Av. Autopista del Saler 16, E-46013, Valencia, Spain. 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.10.012

pharmacokinetics (at the cellular and whole organism level) compared to low molecular weight agents, polymer–drug conjugates have recognised advantages compared to conventional chemotherapy. These include passive tumour targeting due to the enhanced permeability and retention (EPR) effect, a phenomenon that arises due to the hyperpermeability of angiogenic tumour vasculature [6], the ability to reduce toxicity of bound drug [7], and following cellular uptake by the endocytic route, the potential to bypass mechanisms of drug resistance [7,8]. Our (RD) previous studies have given rise to several N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates bearing doxorubicin (Dox), paclitaxel, camptothecin and platinates that have progressed into clinical trial (reviewed in [9,10]). Given the proven lack of clinical toxicity of

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this polymer, more recently conjugates containing experimental drugs (reviewed in [9,11]), and drugs directed towards new therapeutic targets (e.g. anti-angiogenic HPMA copolymer– TNP470 [12]) have emerged. This project is developing the first polymer-based combination therapy for breast cancer. Breast cancer is one of the most common malignancies worldwide and it affects ∼ 1 in 10 women in the UK [13]. Although recent statistics show improved prognosis due to early diagnosis and surgery, often followed by endocrine and/or chemotherapy [15], the outlook is still poor for those patients with metastatic or resistant disease (∼ 20% survival at 5 years). Whereas the selective oestrogen receptor modulator (SERM) tamoxifen was a major advance (28% reduction in mortality of breast cancer patients at 5 years [14]), recent clinical trials have shown that an aromatase inhibitor, anastrozole, is superior to tamoxifen in terms of efficacy, time to recurrence and has less side-effects [15]. Nevertheless, inherent and acquired resistance to both chemotherapy and endocrine therapy still present an unresolved problem [16]. The observations that HPMA copolymer–Dox (FCE28068) showed activity in chemotherapy refractory breast cancer patients in Phase I clinical trials [7], and that aromatase inhibitors can act synergistically with chemotherapy [17], led us to synthesise an HPMA copolymer conjugate containing the aromatase inhibitor aminoglutethimide (AGM) and doxorubicin (Dox) attached to the same polymeric carrier (Fig. 1) [18]. This combination conjugate showed markedly enhanced cytotoxicity against MCF-7 cells in vitro compared to HPMA copolymer–Dox. Furthermore, experiments studying a library of HPMA copolymer conjugates containing AGM alone (the first conjugates to contain endocrine therapy), confirmed aromatase inhibition in vitro, and that AGM liberation was a requirement for activity [18,19]. The aim of this study was to further investigate the mechanism of enhanced cytotoxicity of HPMA copolymer–Dox–AGM, particularly in comparison with HPMA copolymer–Dox. First, further control experiments were conducted to confirm the enhanced cytotoxicity of the polymer combination. It was hypothesised that HPMA copolymer–Dox–AGM and HPMA copolymer–Dox might display (i) different mechanisms or rates of endocytic uptake, (ii) differences in the rate of release of bioactive drug(s) – this has been seen previously with other HPMA copolymer conjugates due to differences in conjugate conformation [20], and/or (iii) differences in the molecular mechanisms of cell killing. The cytotoxicity of conjugates, free Dox and AGM and their mixtures was determined using the MTT assay in MCF-7 and MCF-7ca (aromatase-transfected) cell lines. flow cytometry and live-cell imaging were used to evaluate cell binding (4 °C) and endocytic uptake (37 °C). In addition, studies in the presence of methyl-β-cyclodextrin (MβCD) (inhibits clathrin-mediated and clathrin- and caveolin-independent endocytosis), chlorpromazine (inhibits clathrin-mediated endocytosis) and cytochalasin B (inhibits macropinocytosis) were undertaken to probe the mechanism of endocytic internalisation. The rate of Dox and AGM liberation from the conjugates was measured in vitro in the presence of rat liver lysosomal enzymes (tritosomes) as previously described [21,22]. Finally, immunocytochemistry was used to assess the effect of both conjugates on the

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Fig. 1. Structure and characteristics of drugs tested.

proliferation marker ki67 and the anti-apoptotic protein bcl-2 [23–25]. 2. Materials and methods 2.1. Materials and cells HPMA copolymer precursors carrying –GG–ONp or –GFLG–ONp side chains (either 5 or 10 mol%) of Mw ∼ 20.000–25.000 g/mol and Mw/Mn = 1.3–1.5 were from Polymer Laboratories Ltd. (Shropshire, UK). The ONp content was calculated using ε274 nm in DMSO = 9500 L/mol cm, and the HPMA copolymer–Dox ± AGM and HPMA copolymer–AGM conjugates shown in Fig. 1 were synthesised and characterised as previously described [18]. Anhydrous DMF and optical grade DMSO were from Sigma-Aldrich Company Ltd. (Dorset, UK) and all HPLC grade solvents by Fischer Scientific (Greater Manchester, UK). Medical grade O2, N2 and CO2 (all 95% v/v) and liquid nitrogen were all from BOC Gases (Surrey, UK). AGM was from Aldrich (Dorset, UK) and Dox·HCl was from Pharmacia (Lombardia, Italy). All other reagents were of general laboratory grade and were from Aldrich (Dorset, UK) unless otherwise stated. The human oestrogen-dependant, breast carcinoma cell lines MCF-7 and MCF-7ca (human aromatase-transfected) were from the Tenovus Centre for Cancer Research at Cardiff University. Cells were cultured in RPMI 1640 with L-glutamine medium supplemented with 5% of foetal bovine serum (FBS) as standard tissue culture conditions. RPMI and FBS were from

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GIBCO BRL Life Technologies (Paisley, UK). In order to maintain the transfected strain, the culture medium of MCF-7ca was always (for routine tissue culture and for all the experiments) further supplemented with 0.75 mg/mL of geneticin (supplied by Fluka, Dorset, UK). Tissue culture grade DMSO, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Trypan blue solution (0.4%) (cell culture grade) and oestradiol were from Sigma (Dorset, UK). Steroid-deprived FCS (SFCS) was prepared as described previously [19]. For immunocytochemistry, the solvents used for fixation (methanol and acetone) and formaldehyde solution (40%) were from Fisher Scientific (Leicestershire, UK). The monoclonal mouse primary antibodies against Ki67 antigen (clone MIB-1), and bcl-2 oncoprotein (clone 124), polyclonal goat anti-mouse (GAM) immunoglobulins, peroxidase anti-peroxidase (PAP), and diaminobenzidine (DAB) were all from DakoCytomation (CA, USA). The supersensitive concentrated detection kit (i.e. Link (biotinylated anti-mouse immunoglobulins) and the concentrated label (streptavidin peroxidase) were from Biogenex (CA, USA). The DPX mountant used for histology, Tween and the methyl green were all from Sigma-Aldrich (Dorset, UK). Glycerol and sucrose were from Fisher Scientific (Leicestershire, UK). 2.2. Cytotoxicity of Dox, AGM and HPMA copolymer conjugates The cytotoxicity of free drugs (AGM and Dox) and their conjugates was evaluated using the MTT cell viability assay (72 h of incubation) with MCF-7 and MCF-7ca cells. Both cell lines were seeded into sterile 96-well microtitre plates (4 × 104 cells/mL) in WRPMI 1640 with 5.0 mM L-glutamine and 5% (v/v) of SFCS ± 10− 9 M oestradiol. They were incubated for 5 days before the test compounds (0.2 μm filtersterilised) were added to give a final concentration in the range of 0–2 mg/mL drug-equiv. After a further 67-h incubation, MTT (20 μL of a 5 mg/mL solution in PBS) was added to each well, and the cells were incubated for 5 h. After removal of the medium, the precipitated formazan crystals were dissolved in optical grade DMSO (100 μL), and the plates were read spectrophotometrically at 550 nm after 30 min using a microtitre plate reader. Cell viability was expressed as a percentage of the viability of untreated control cells. The IC50 values were expressed as concentration (μg/mL) of AGM or Dox equiv. 2.3. Binding and uptake of HPMA copolymer–Dox ± AGM conjugates by MCF-7 and MCF-7 cells MCF-7 cells were seeded in 6-well plates at a density of 1 × 106 cells/mL (1 mL cell suspension per well) and allowed to adhere for 24 h. In the binding experiments conducted at 4 °C, the cells were pre-incubated at this temperature for 30 min prior to addition of conjugate. HPMA copolymer–Dox conjugate (5) and HPMA copolymer–Dox–AGM conjugate (4) (both 6.3 μg/mL Dox-equiv.) were added and the cells incubated for 0 to 60 min either at 37 °C or 4 °C. Note that for 0 min incubation, the conjugate was added to the cells and immediately removed. At each sample time, cells were placed on ice, then washed

three times with ice-chilled PBS (5 mL), and PBS (1 mL) was added before the cells were scraped from the plate with a rubber policeman and collected in falcon tubes. The cell suspension was centrifuged at 4 °C (600×g for 10 min). The cell pellet was re-suspended in ice-chilled PBS (200 μL). Cell-associated fluorescence was then analysed using a Becton Dickinson FACSCalibur cytometer (CA, USA) equipped with an argon laser (488 nm) and emission filter for 550 nm. Data collection involved 25,000 counts per sample, and the data were analysed using CELLQuest™ version 3.3 software. Data are expressed by plotting the shift in geometric mean of the entire population (i.e. geometric mean after incubation with the polymer − geometric mean in absence of polymer). Cells incubated without polymer conjugate were used to account for the background fluorescence. 2.4. Evaluation of the cytotoxicity of endocytosis inhibitors Before using the endocytosis inhibitors it was important to define their general toxicity. MCF-7 and MCF-7ca were seeded in 24 well plates (3.2 × 105 cells/mL; 500 μL per well) in WRPMI + 5% SFCS. The seeding density was adjusted to obtain the same concentration (cells/area) used in the flow cytometry experiments. After 24 h, cells were washed with warm (37 °C) PBS (500 μL) to remove dead cells and residual serum. The inhibitors were added in medium (500 μL) using the following concentrations: a combination of MβCD (0–15 mM) and lovastatin (1 μM); chlorpromazine (0–50 μM); or cytochalasin B (0–25 μM). After 2 h, the medium was removed, the cells were washed with warm (37 °C) PBS (3 × 500 μL), and then, 150 μL of a Trypan blue solution (0.2% Trypan blue in PBS) was added to each well. The total number of stained cells in the visual field was counted (SCvis). The total number of cells in the visual field (TCvis, i.e. stained and unstained) was determined applying a grid that allowed the counting of 25% of the cells in the visual field. The data were expressed as % of dead cells as follows: percentage dead cells ¼ ðSCvis =TCÞ  100: 2.5. Evaluation of cellular uptake in presence of inhibitors of endocytic pathways MCF-7 and MCF-7ca were seeded as described above in 6well plates (1 × 106 cells/mL). After 24 h, the cells were washed with warm (37 °C) PBS and fresh medium (900 μL) was added that contained the endocytosis inhibitor; MβCD + lovastatin, chlorpromazine or cytochalasin B. The solutions were prepared starting from stock solutions of the inhibitor (50 mg/mL and 10 mg/mL, 10 mg/mL or 2 mg/mL, respectively). The final concentrations used were 10 mM + 1 μM for MβCD + lovastatin, 25 μM for cytochalasin B and 15 μM for chlorpromazine, respectively. In each case it was ensured that solvents were also used at non-toxic concentrations. After a 1-h pre-incubation with the inhibitors at 37 °C, HPMA copolymer–Dox (5) or HPMA copolymer–Dox–AGM (4) (100 μL in medium) was added (final concentration 6.3 μg/mL Dox-equiv.). After 1 h the cells were placed on ice, and prepared for FACS as described above.

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2.6. Confocal fluorescent microscopy: live-cell imaging For live-cell imaging, MCF-7 and MCF-7ca cells (1 × 106 cells) were seeded in glass bottom culture dishes and left to adhere to the coverslips for N 12 h. To assess the subcellular localisation of the polymers, cells were incubated for either 15 min or 1 h at 37 °C in complete medium (1 mL) containing HPMA copolymer–Dox ± AGM conjugates (6.3 μg/mL Doxequiv.). Before visualisation, medium containing the polymers was removed from the cells by aspiration, and the cells were washed three times with fresh medium (37 °C). Finally, clear complete media was added and cells were subsequently viewed for a maximum of 30 min. Images were captured with an inverted DM IRE2 microscope equipped with a λ-blue 60× oil immersion objective and handled with a TCS SP2 system, equipped with an Acoustic Optical Beam Splitter (AOBS). Excitation was with an argon laser (548 nm, 476 nm, 488 nm, 496 nm and 514 nm) and blue diode (405 nm). Images were captured at an 8-bit grey scale and processed with LCS software (version 2.5.1347a, Leica Germany) containing multicolour, macro and 3D components. 2.7. Release of AGM and Dox from HPMA copolymer conjugates during incubation with isolated rat liver lysosomal enzymes Tritosomes were prepared in sucrose according to the method of Trouet [21]. Protein content was determined using bicinchoninic acid protein assay (BCA) and proteolytic activity was determined by measuring the release of p-nitroanilide (NAp) from N-benzoyl-Phe-Val-Arg–NAp. In this case, the tritosomes used had a protein content of 2.3 mg/mL and an activity of 62.4 nm/min/mg protein. HPMA copolymer–Dox ± AGM conjugates (50 μg/mL drug-equiv.) were incubated (37 °C) in citrate phosphate buffer (pH 5.5, 1 mL total volume) with Triton X-100 (0.2% w/v), EDTA (100 μL, 10 mM) and GSH (100 μL, 50 mM) [22]. To begin the degradation study tritosomes were added (300 μL) and the Eppendorf tubes thoroughly mixed. Aliquots (100 μL) were taken at times up to 24 h, immediately frozen in liquid nitrogen, and stored frozen in the dark until assayed by HPLC. In control experiments conjugates were incubated in buffer alone (without addition of tritosomes) to assess non-enzymatic hydrolytic cleavage. In addition, free drug (1 or 2) (50 μg/mL) was also incubated under same conditions and later used as the reference control in the HPLC assay. 2.7.1. Determination of AGM or Dox release by HPLC Samples were defrosted and added to polypropylene tubes and made up to 1 mL with water. The pH of the samples was adjusted to 8.5 with ammonium formate buffer (100 μL, 1 M, pH 8.5), and then a mixture of chloroform/2-propanol at a ratio of 4:1 (5 mL) was added. Dnm was used as internal reference standard; 100 μL of a 1 μg/mL stock aqueous solution was added to each sample. Samples were then thoroughly extracted by vortexing (3 × 10 s). The upper aqueous layer was carefully removed and the solvent was evaporated under N2. The dry residue was dissolved in 100 μL of HPLC grade methanol. In

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parallel, the same procedure was carried out for the parent compounds 1 and 2 (using 100 μL of a 1 mg/mL stock aqueous solution) to construct a standard curve. Addition of 1 mL of methanol to redissolve the product gave a 100 μg/mL stock from which a range of concentrations were prepared (2 to 60 μg/ mL). The amount of drug released from the conjugates was determined by HPLC using a μBondapak C18 column (150 × 3.9 mm), with a flow rate of 1 mL/min and using a gradient elution [solvent A: 2-propanol/H2O 12:88 (v/v), solvent B: 2-propanol/H2O 29:71 (v/v)] adjusted to pH 3.2 with o-phosphoric acid. Total run time was 20 min and the gradient profile was: t = 5 min A 100%, t = 9 min A 0%, t = 14 min A 0%, t = 16 min A 50%, t = 18 min A 100% and t = 20 min A 100%). To monitor AGM (1) an UV detector (Spectroflow 783 Kratos analytical) with a fixed-wavelength filter (254 nm) was used. To monitor Dox (2) and the Dnm standard a Fluoromonitor III fitted with interference filters at 485 nm for excitation and 560 nm for emission was used. The retention time was 4.67 min for AGM and 12.48 min for Dox. 2.8. Investigating the ability of AGM to inhibit tritosomes and cathepsin B The activity of tritosomes and cathepsin B against the Nbenzoyl-Phe-Val-Arg–NAp substrate were determined by UV as described above. To study the effect of AGM on enzyme activity, AGM (25–200 μg/mL in DMSO, 10 μL) was added to the incubation mixture. Addition of DMSO alone (10 μL) was used as a control. A known inhibitor of the thiol-dependent proteases, leupeptin (50 μg/mL) was used as a positive control. As above, the tritosomes or cathepsin B were warmed to 37 °C (30 μL) and added last to start the reaction. Release of NAp at 410 nm was measured by UV over 20 min. 2.9. Immunocytochemistry The expression of the proliferation protein ki67 and of the anti-apoptotic protein bcl-2 was evaluated by immunocytochemistry in MCF-7 and MCF-7ca cells in the presence and absence of drugs and conjugates. The experimental protocol was designed to resemble, as closely as possible, the experimental conditions used for the determination of conjugate cytotoxicity as described above. Both cell lines were seeded in sterile 12-well plates that contained one sterile tespa-coated round coverslip (16 mm diameter) per well in WRPMI supplemented with 5% of SFCS. The seeding density was 1.66 × 105 cells/mL (0.5 mL/well) and the cells were allowed to adhere for 24 h. Then, the medium was replaced with fresh medium that contained 5% SFCS ± 10− 9 M oestradiol. After 5 days the medium was replaced with fresh medium that contained either HPMA copolymer–Dox–AGM or HPMA copolymer–Dox (0.2 μm filter sterilized) at a Dox concentration that corresponded to the IC50 values determined for the HPMA copolymer–Dox–AGM (IC50 in MCF-7 75 or 21 μg/mL Dox-equiv. with or without oestradiol, respectively; in MCF-7ca 8.2 or 12 μg/mL Dox-equiv. with or without oestradiol, respectively).

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On day 8 (after 72 h of incubation with the polymer conjugates), the cells were fixed as follows. The coverslips were placed in formaldehyde solution (4% in PBS) for 15 min at room temperature, then into PBS for 5 min room temperature. Subsequently, they were transferred to methanol (5 min) and acetone (3 min) which were kept on dry ice to ensure that the temperature of the solvents was maintained between − 30 °C and − 10 °C. Finally there was a PBS wash (5 min at room temperature) and the coverslips were stored in a sucrose storage solution (42.8 g sucrose, 0.33 g magnesium chloride anhydrous, 250 mL PBS and 250 mL of glycerol) at − 20 °C. 2.9.1. Detection of Ki67 The storage medium was removed by thorough washings with PBS followed by a wash with PBS–Tween (0.02%). Then, the solution containing the primary antibody (against Ki67 antigen, clone MIB-1) (1:75 in PBS) was added (30 μL per coverslip) before incubation at room temperature for 60 min. Then, the primary antibody was removed by repeated washing with PBS– Tween and the secondary antibody added (Biogenex mouse link 1:50 in BSA–PBS (1%); 50 μL per coverslip). After 30 min at room temperature, the coverslip was washed with PBS–Tween and the tertiary antibody was added (Biogenex Concentrated Label dilution 1:50 in BSA–PBS (1%); 50 μL per coverslip) followed by a further 30 min incubation at room temperature. Finally, the coverslips were washed with PBS–Tween and incubated with the peroxidase substrate and DAB for 10 min. After thorough washing with dH2O and the cells were counterstained with a solution of methyl green (0.5% in dH2O) for 30 s at room temperature, washed again with dH2O and allowed to air dry before mounting onto a glass slide using DPX mountant. 2.9.2. Detection of bcl-2 The storage medium was removed by thorough washings with PBS followed by a wash with PBS–Tween (0.02%). Then, a solution of normal goat serum (5%) and normal human serum (5%) in PBS was added (50 μL/coverslip) and after 10 min the excess was then removed. The primary antibody (against bcl-2 oncoprotein, clone 124) solution was prepared pre-absorbing 1 μL of primary antibody with 2 μL of normal human serum for 90 min and then adding 747 μL of 0.1% BSA in PBS. The primary antibody was added (30 μL/coverslip) and left overnight before repeated washing with PBS–Tween. Then the secondary antibody, goat–anti-mouse (1:35 in PBS; 50 μL/coverslip) was added and left for 30 min before another wash with PBS–Tween, addition of the tertiary antibody (peroxidase anti–peroxidase; 1:250 in PBS; 50 μL/coverslip), and left for another 30-min incubation. Then the coverslips were washed with PBS–Tween, and a solution containing the peroxidase substrate and DAB was added and left for 10 min. Finally, the coverslips were washed with dH2O, and the cells were counterstained with a solution of methyl green (0.5% in dH2O) for 30 s at room temperature. After a final wash with dH2O they were allowed to air dry before being mounted onto a glass slide. In each case the immunostaining was assessed by two independent observers. Data are expressed as a percentage of cells with positive staining.

2.10. Statistical analysis When only two groups were compared, the Student's t test for small sample size was used to estimate statistical significance. If more than two groups were compared evaluation of significance was performed using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. In all cases, statistical significance was set at p b 0.05. 3. Results The synthesis of the HPMA copolymer conjugates (3, 4, 5) has been described elsewhere [18,19], but the characteristics of the specific samples prepared for these studies are given in Fig. 1. Earlier studies using MCF-7 and MCF-7ca cells clearly showed an enhancement of cytotoxicity in vitro when AGM and Dox were conjugated within the same polymeric backbone compared to the effects seen when HPMA copolymer–Dox or HPMA copolymer–AGM (or a simple mixture of both were

Fig. 2. Comparison of the cytotoxicity HPMA copolymer–Dox–AGM and conjugate mixtures in MCF-7 and MCF-7ca cells. Panel (a) shows cytotoxicity in MCF-7; panel (b) shows cytotoxicity in MCF-7ca; panel (c) the aromatase expression in both cell lines. n, Dox; □, HPMA copolymer–Dox–AGM; ●, HPMA copolymer–Dox + HPMA copolymer–AGM; ○, HPMA copolymer– Dox. Data are represented as mean ± S.E.M.; n = 3.

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The cytotoxicity of all free drugs, HPMA copolymer conjugates and their mixtures against MCF-7 and MCF-7ca cells cultured in the presence and absence of oestradiol (to see if the cytotoxicity of these drugs was affected by the presence of this hormone) is shown in Fig. 3 and Table 1). Free AGM and HPMA copolymer–AGM showed little cytotoxicity in either cell line under the experimental conditions used. As expected, Dox was cytotoxic, being ∼ 55-fold more active in MCF-7 than MCF-7ca cells. Addition of Dox + AGM did not change the IC50 values obtained when expressed as Dox-equiv. indicating no synergistic effect. Neither addition of AGM (1), nor HPMA copolymer–AGM conjugate (3) to HPMA copolymer–Dox (5) caused an increase in cytotoxicity compared to the profile seen for the individual compounds (Fig. 3). For example, in MCF7ca cells the IC50 value of 1 + 5 was 967 μg/mL (AGM-equiv.) compared to 1043 μg/mL for (1) alone. HPMA copolymer– Dox–AGM (4) displayed greater activity than HPMA copolymer–Dox (5), and this enhanced activity was most evident in MCF-7ca cells where the IC50 value seen (Dox-equiv.) was similar to that seen for free Dox (Table 1). 3.1. Comparison of the endocytic properties of HPMA copolymer–Dox–AGM and HPMA copolymer–Dox Prior to flow cytometry studies, the fluorescence characteristics of HPMA copolymer–Dox (5) and HPMA copolymer– Dox–AGM (4) were determined to ensure that correct comparisons would be made. Although the fluorescence of neither conjugate was affected by pH, HPMA copolymer–Dox had a higher fluorescence output (∼ 1.3- to 1.4-fold) than conjugate (4) across the Dox concentration range studied (Fig. 4a).

Table 1 Cytotoxicity of Dox and AGM and HPMA copolymer conjugates against MCF-7 and MCF-7ca human breast cancer cell lines a Entry Compound(s)

Fig. 3. Cytotoxicity of Dox, AGM and HPMA copolymer conjugates and their mixtures in MCF-7 and MCF-7ca cells incubated in the presence and absence of oestradiol. n, MCF-7 plus oestradiol; □, MCF-7 without oestradiol; ●, MCF-7ca plus oestradiol; ○, MCF-7ca without oestradiol. Data are represented as mean ± S.E.M.; n = 3.

added (preliminary supplementary information in [18]). Further experiments reported here confirm the uniqueness of this effect (Figs. 2 and 3, and Table 1). The HPMA copolymer–Dox– AGM conjugate (4) was more toxic in both MCF-7 and MCF7ca cells than HPMA copolymer–Dox (5) and a mixture of HPMA copolymer–Dox (5) + HPMA copolymer–AGM (3) (Fig. 2), and activity was similar to free Dox in MCF-7ca cells, an effect which correlates with the higher aromatase levels in these cells.

1 2 3 4 5 6

7

8

AGM ( 1) Dox ( 2) Dox ( 2) + AGM ( 1) HPMA copolymer– AGM ( 3) HPMA copolymer– Dox ( 5) HPMA copolymer– Dox ( 5) + AGM ( 1) HPMA copolymer– Dox ( 5) + HPMA copolymer–AGM ( 3) HPMA copolymer– Dox–AGM ( 4)

Dox (IC50) b

AGM (IC50) c

MCF-7

MCF-7ca MCF-7

MCF-7ca

NA 0.4 ± 0.2 0.4 ± 0.1 NA

NA 22 ± 8.5 16 ± 7.6 NA

1223 ± 274 NA 7.4 ± 1.7 N180 d

1043 ± 441 NA 308 ± 151 N180 d

N126 d

N126 d

NA

NA

62 ± 12

60 ± 8.9

988 ± 177

967 ± 145

N157 d

N157 d

N151 d

N151 d

75 ± 45

8.2 ± 3.1

77 ± 45

8.8 ± 3.5

a Cell viability 72 h MTT assay, seeding density 4 × 104 cells/mL for the experiments carried out in the presence of 10− 9 M oestradiol in medium. Published as supplementary information in Vicent et al. [18]. b Data expressed in μg/mL of Dox-equiv. mean ± S.E.M.; n = 3. c Data expressed in μg/mL of AGM-equiv. mean ± S.E.M.; n = 3. d Maximum concentration tested.

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Addition of HPMA copolymer conjugates to both MCF-7 and MCF-7ca cells led to an immediate fluorescence shift that progressed further during the 1 h incubation (Fig. 4b). Both conjugates (3 and 4), in both cell lines, showed higher cellassociated fluorescence at 37 °C than at 4 °C (Fig. 4c,d). Although the initial increase in cell association was rapid (within minutes), there was a much lower time-dependent increase thereafter (10–60 min) (Fig. 4c,d). Incubation with HPMA copolymer–Dox (5) resulted in higher cell-associated fluorescence than was observed for the combination conjugate (4) at both temperatures and in both cell lines, but this have been partially attributed to the higher fluorescence output of the conjugate. Live-cell imaging revealed most fluorescence associated to the plasma membrane at 15 min (data not shown), and in agreement with the flow cytometry studies, this was more marked for conjugate (5) than (4). Even at 15 min, it was possible to see HPMA copolymer–Dox–AGM (4) in vesicles within MCF-7 cells, and these were more numerous and prominent than could be seen for (5). After 1 h, vesicular labelling was much clearer for both conjugates, and similar observations could be seen with the MCF-7ca cell line (Fig. 5). A number of inhibitors of endocytosis were selected to investigate whether conjugates (4) and (5) would demonstrate distinct mechanisms of uptake. It is necessary to take care that such inhibitors are always used at non-toxic concentrations. In MCF-7 cells, all the compounds showed a dose-dependent toxicity (Fig. 6a,b). MβCD caused less than 20% cell death at concentrations ≤15 mM. Cytochalasin B toxicity never caused N10% cell death, even at the maximum concentration tested (25 μM), and chlorpromazine caused ∼ 100% cell death at

Fig. 4. Fluorescence characteristics of HPMA copolymer conjugates and their cell binding and endocytic uptake. Panel (a) shows the pH and concentration dependence of Dox fluorescence in conjugates 4 and 5. Panel (b) shows representative data in flow cytometry experiments collected after incubation of MCF-7ca cells with HPMA copolymer–Dox. Panels (c) and (d) show timedependent binding (4 °C) and uptake (37 °C) of HPMA copolymer–Dox and HPMA copolymer–Dox–AGM by MCF-7 and MCF-7ca cells, respectively. □, HPMA copolymer–Dox at 37 °C; ○, HPMA copolymer–Dox at 4 °C; n, HPMA copolymer–Dox–AGM at 37 °C; ●, HPMA copolymer–Dox–AGM at 4 °C. Inserts show magnification of the data obtained with the combination conjugate. Data are represented as mean ± S.E.M.; n ≥ 3. ⁎p b 0.05.

Flow cytometry and confocal microscopy experiments conducted at equi-concentrations of Dox-equiv. should always be interpreted with this in mind.

Fig. 5. Live-cell confocal microscopy images of MCF-7 and MCF-7ca cells incubated (37 °C) with conjugates (4) and (5). In each case a conjugate concentration of 6.3 μg/mL Dox-equiv. was used. The white arrows indicate membrane binding, blue arrows indicate vesicular localisation. Scale bar = 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

F. Greco et al. / Journal of Controlled Release 117 (2007) 28–39

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50 μM. Results obtained in MCF-7ca cells showed a similar pattern of toxicity, and again chlorpromazine was the most toxic (∼ 30% cell killing at 50 μM). As the inhibitor concentrations commonly used in the literature are 10 mM for MβCD, 15 μM for chlorpromazine and 25 μM for cytochalasin B (all caused ≤ 10% cell killing) these concentrations were adopted for further studies. After a 1 h incubation, MβCD decreased the uptake of both HPMA copolymer–Dox (5) and HPMA copolymer–Dox– AGM (4) in MCF-7 by ∼ 60% and ∼ 50%, respectively. A similar trend (although at a lower extent) was seen in MCF-7ca were the uptake of both conjugates was decreased by ∼25%. Incubation with chlorpromazine or cytochalasin B decreased only the uptake of HPMA copolymer–Dox (5) in MCF-7 (∼ 50% and ∼ 20%, respectively) but did not have any effect on the uptake of the other conjugate or in MCF-7ca. Overall, a similar response to the presence of these inhibitors was seen for both cell lines and for both conjugates. Chlorpromazine inhibited HPMA copolymer–Dox (∼ 60%) uptake in MCF-7 cells. However, the inhibitors used (at these concentrations) also had a similar effect on both conjugates (4) and (5). Fig. 7. Drug release from HPMA copolymer conjugates on incubation with rat liver lysosomal enzymes (tritosomes). Panel (a) shows release of Dox from conjugate (5) in the presence and absence of conjugate (3); panel (b) shows the release of AGM from conjugate (3) in the presence and absence of conjugate (5), and panel (c) shows both Dox and AGM release from the HPMA copolymer– GFLG–Dox–AGM conjugate (4). In all cases, drug release is expressed as a percentage of the total drug bound, and data are represented as mean ± S.E.M. (n = 4). The release of Dox from HPMA copolymer–Dox (panel a) and the release profiles reported in panel (c) have been published previously in Vicent et al. [18].

3.2. Drug release from HPMA copolymer conjugates

Fig. 6. Cytotoxicity of inhibitors of endocytosis and their effect (at non-toxic concentrations) on the uptake of HPMA copolymer conjugates (4) and (5) by MCF-7 and MCF-7ca cells. Panels (a) and (b) show inhibitor concentrationdependent toxicity. Panels (c) to (f) show the effect of inhibitors on the uptake of conjugate (6.3 μg/mL Dox-equiv.) when cells were incubated for 1 h. n, Control; □, MβCD; , chlorpromazine; , cathepsin B. Data are represented as mean ± S.E.M., n = 6. ⁎p b 0.05.

As anticipated, tritosomes released AGM and Dox from all the HPMA copolymer conjugates containing the Gly-Phe-LeuGly polymer–drug linker. No degradation occurred in the absence of enzymes (e.g. Fig. 7b). The drug-release profile displayed marked differences depending on conjugate composition. The combination conjugate HPMA copolymer–Dox– AGM released between 15–20% AGM and ∼ 20% of Dox over 5 h, and unexpectedly release was not linear with time (Fig. 7c). Release of both Dox and AGM had a lag phase with little release over the first 30 min, but after this time AGM initial release (to a plateau) was much faster than Dox release which caught up over 5 h. To understand the reason for these unusual kinetics, the release of Dox from conjugate (5) was measured in the presence and absence of HPMA copolymer–AGM (3) (Fig. 7a). In both cases, Dox release began immediately after addition of enzyme and was linear with time. Although the presence of (3) reduced the extent of Dox release this was not surprising due to increase of effective substrate concentration. The release of AGM from conjugate (3) was also measured in the presence of conjugate (5). In both cases, AGM was initially released quickly, but after ∼200 min started to plateau. The reason for this behaviour is not clear as active enzyme is available over the whole

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incubation period. One explanation could be that the AGM released causes enzyme inhibition. To test this hypothesis, cathepsin B and tritosomes were incubated with Bz–ValArgPhe–NAp as substrate in the presence and absence of AGM, using the inhibitor leupeptin as positive control. Studies on the kinetics of degradation by tritosomes gave a Vmax = 0.107 Abs/min and Km = 0.3 μM. Addition of AGM (a concentration 50 μg/mL corresponds to the maximum release of AGM in Fig. 8b,c) caused ∼30–40% inhibition of tritosome activity, but only 4–8% inhibition of cathepsin B activity (Table 2). AGM inhibition was not dependent on concentration, and lower than that observed for leupeptin.

Table 2 AGM inhibition of tritosomes and cathepsin B AGM (μg/mL)

0 25 50 200 Leupeptin (50 μg/mL) a b

Tritosomes

Cathepsin B

Activity a (nM/min/mg)

Inhibition a (%)

Activity b (nM/min/mg)

Inhibition b (%)

37 24 21 25 0

0 34 42 32 100

91 ± 12 75 ± 5 78 ± 8 78 ± 10 0±0

0±0 8±7 4±3 4±5 100 ± 0

Data expressed as mean of n = 2 experiments. Data expressed as mean ± S.E.M. (n = 3).

3.3. Immunocytochemistry To investigate whether HPMA copolymer–Dox–AGM (4) and HPMA copolymer–Dox (5) had different effects on cell proliferation, immunocytochemistry was used to study ki67 and

Fig. 8. Immunocytochemistry showing the effect of HPMA copolymer conjugates (4) and (5) on the expression of ki67 in MCF-7 and MCF-7ca cells. Cells were incubated in the presence of oestradiol as shown. The black arrows indicate nuclear staining, red arrows nucleoli staining and white arrows unstained cells. Scale bar = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

bcl-2 expression in MCF-7 and MCF-7ca cells following incubation with each conjugate. In MCF-7ca cells, HPMA copolymer–Dox caused a decreased in expression of ki67 (Fig. 8b), and HPMA copolymer–Dox–AGM decreased expression even further (Figs. 8c and 9). Similar trends were seen in MCF-7 cells (Figs. 8d–f and 9), and also in absence of oestradiol (Fig. 9). Levels of bcl-2 were used to assess the effect of both two conjugates (4 and 5) on apoptosis. There was no difference in expression in MCF-7 cells incubated with or without HPMA copolymer–Dox (Fig. 10). However, MCF-7 cells incubated with HPMA copolymer–Dox–AGM showed decreased expression of bcl-2 (∼30% reduction) (Fig. 10c,d). A similar trend was found in MCF-7 cells in absence of oestradiol (Fig. 10d). Assessment of bcl-2 in MCF-7ca led to poor staining (b 10%) in

Fig. 9. Expression of ki67 in MCF-7 and MCF-7ca cells. □, Control; n, control+ oestradiol; , HPMA copolymer–Dox; , HPMA copolymer–Dox–AGM. Note that the set of results on the left was performed in absence of oestradiol, while the set of results on the right was performed in presence of oestradiol (10− 9 M). Data are represented as mean ± S.E.M.; n ≥ 8. ⁎p b 0.05.

F. Greco et al. / Journal of Controlled Release 117 (2007) 28–39

Fig. 10. Effect of HPMA copolymer conjugates (4) and (5) on the expression of bcl-2 in MCF-7 cells. Cells were incubated in the presence or absence of oestradiol as shown. The key for panel (d) is as shown in Fig. 9. The values in panel (d) are represented as mean ± S.E.M.; n ≥ 6. ⁎p b 0.05.

all cases (data not shown), therefore investigation of the impact of the conjugates on this marker was impossible for this cell line.

37

antitumour activity in heavily pretreated (even anthracyclineresistant) breast cancer patients [7]. The inability of AGM + Dox, and all other permutations of non covalent mixtures of drugs and conjugates studied here to show synergistic effects in the MCF-7 cell lines underlines the uniqueness of the combination conjugate. Hypothetical opportunities for differences in the mechanism of action of HPMA copolymer–Dox and HPMA copolymer–Dox–AGM conjugate are summarised schematically in Fig. 11. Although direct comparison of binding and uptake of conjugates 4 and 5 proved more challenging than expected due to their differences in fluorescence yield (Fig. 4a), it is clear that both conjugates displayed the same pattern of cell association. If anything, HPMA copolymer–Dox–AGM was captured more slowly (Fig. 4c,d) and certainly not fast enough to account for the marked increase in cytotoxicity compared to HPMA copolymer–Dox (Fig. 2). Surprisingly in the MCF-7 cell lines both conjugates showed very high binding. Although live-cell confocal microscopy confirmed vesicular uptake after 15 min, the internalisation measured by FACS was relatively slow thereafter. Inhibitors of endocytic entry pathways were used to see whether the conjugates were using distinctly different entry portals. Reduced uptake of both conjugates 4 and 5 seen in presence of MβCD (Fig. 6) suggested, at least in part, internalisation via a cholesterol-dependent pathway [31]. Clathrin- and caveolin-dependent uptake require cholesterol [32–34], but as wild type MCF-7 cells do not express a detectable amount of caveolin [35] this uptake may be via coated vesicle pathway. Further studies with chlorpromazine (this compound is claimed to inhibit only clathrin-dependent uptake [31,36,37]) resulted in some inhibition of uptake (but less than

4. Discussion The last decade has seen significant advances in the management of breast cancer that are attributable to earlier diagnosis, the emergence of endocrine therapy (e.g. tamoxifen and aromatase inhibitors), and most recently the introduction of Herceptin®. However, inherent and acquired resistance is still a significant problem. It is increasingly clear that monotherapy is unlikely to provide a long-term solution for the treatment of metastatic and/or resistant disease [26]. Conventional chemotherapy is routinely used as combination therapy and recent studies are showing the potential of polymer–protein [27] and polymer– drug conjugates when combined with another chemotherapeutic agent [28,29]. Furthermore, HPMA copolymer conjugates carrying both chemotherapy (Dox) and phototherapy have been described that have increased antitumour activity against OVCAR-3 xenografts in mice [30]. A polymeric carrier provides an ideal platform for the simultaneous delivery of a cocktail of drugs, and we recently reported the first endocrine–chemotherapy combination in the form of the model compound HPMA copolymer–Dox–AGM (4) [18]. Although this conjugate displayed markedly enhanced cytotoxicity compared to HPMA copolymer–Dox (5), its precise mechanism of enhanced activity remains unclear. AGM released from the polymer conjugate can inhibit aromatase in MCF-7 cells [19], and HPMA copolymer–Dox can exhibit clinical

Fig. 11. Proposed mechanism of action of the HPMA copolymer–Dox–AGM.

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seen for MβCD). Cytochalasin B only caused significant inhibition of HPMA copolymer–Dox in MCF-7 cells but the extent of inhibition was relatively small. All that can be concluded from these studies is that conjugates 4 and 5 did not show marked differences in uptake mechanism. Both 4 and 5 were designed using a Gly-Phe-Leu-Gly linker to ensure lysosomotropic Dox delivery [38]. It has been shown previously that linker degradation by thiol-dependent proteases (particularly cathepsin B) are a prerequisite for HPMA copolymer conjugate activity (reviewed in [9,19]). The lysosomal enzyme-mediated drug release profiles for conjugates 3, 4 and 5 were strikingly different (Fig. 7). Failure of either conjugate (3 and 5) to modify the drug release profile of the other when simply added to the mixture (Fig. 7a,b) underlines the uniqueness of the kinetics of release observed for the combination conjugate 4. Differences in structure (or solution dynamics) of the complex unimolecular micelles that are 3, 4 and 5 could theoretically be responsible for the release pattern observed. The conjugates certainly have different Rg as defined by SANS (Fig. 1) [18] and the structural geometry of each may guide enzyme access, to the monomers bearing drug (either AGM or Dox). Another physicochemical factor that could theoretically influence enzyme accessibility would be “blockyness” in structure, with polymer domains containing a high density of pendant drug molecules. Theory suggests this should not be the case as the HPMA copolymer precursor is a random copolymerisation of HPMA monomer and monomer containing the Gly-Phe-Leu-Gly–ONp side chain. Subsequent drug conjugation should also give a statistical distribution along the polymer chain. However, this cannot be guaranteed in practice and further studies using block copolymers especially prepared with an AGM-containing block and a Dox-containing block would help to elucidate further this question. The rather strange kinetics of AGM release in both 3 and 4 (drug liberation reaching a plateau) might theoretically be explained by AGM– enzyme inhibition. However, AGM showed little inhibition of cathepsin B, and although it did inhibit tritosome activity this was not concentration-dependent (Table 2). Although HPMA copolymer–Dox was synthesised more than 20 years ago [39] and its activity is well established both in animal models [40] and in clinical setting (reviewed in [9,10]), its precise mechanism of action at cellular level is still unclear [4,10]. This is not unusual for many anticancer agents. The preliminary immunocytochemical studies (Figs. 8 and 9) showed decreased expression of ki67 following incubation of MCF-7 and MCF-7ca cells with conjugates 4 and 5. This was most marked for the combination polymer. As ki67 is a well described marker for the proliferating fraction of a cell population [23] this is consistent with the higher cytotoxicity of 4. Bcl-2 expression following incubation of various cell types with HPMA copolymer–Dox has been investigated before, but the results obtained are conflicting. Some studies suggest decrease in bcl-2 expression [41,42] while others did not see any effect on the level of this protein [43]. The MCF-7 results reported here agree with the latter study as 5 had no effect on bcl-2. Decreased bcl-2 was however seen following incubation of MCF-7 with conjugate 4 suggesting that combined AGM and

Dox leads to a synergistic effect that induces apoptosis, hence the increased activity of the combination polymer. 5. Conclusions Complex cellular mechanisms seem to be responsible for the increased antitumour activity of HPMA copolymer–Dox– AGM has in vitro. Differences in the drug release profile were evident and this conjugate caused a significant change in the expression of the anti-apoptotic protein bcl-2. Further studies are needed to investigate these effects further, but it is likely that in vivo testing will be required to better define both therapeutic potential of HPMA copolymer–Dox–AGM conjugate and the exact mechanism of action. Acknowledgements F.G. and M.V. thank the Centre for Polymer Therapeutics, Tenovus Centre for Cancer Research, and Marie Curie (HPMFCT-2002-01555) for supporting their work. The authors would also like to thank Pauline Finlay, Sue Kyme, Iain Hutcheson and Jan Knowlden for advice regarding immunocytochemistry and Western blotting techniques. References [1] R. Duncan, The dawning era of polymer therapeutics, Nat. Rev. Drug Discov. 2 (2003) 347–360. [2] R. Satchi-Fainaro, R. Duncan, C.M. Barnes, Polymer therapeutics for cancer: current status and future challenges, Adv. Polym. Sci. 193 (2006) 1–65. [3] M.J. Vicent, R. Duncan, Polymer-based nanomedicines: novel treatments for cancer, Trends Biotechnol. 24 (2006) 39–47. [4] R. Duncan, Polymer–drug conjugates as anticancer nanomedicines, Nat. Rev. Cancer. 6 (2006) 688–701. [5] J.W. Singer, S. Schaffer, B. Baker, A. Bernareggi, S. Stromatt, D. Niensted, M. Besman, Paclitaxel poliglumex (XYOTAX; CT-2103) [XYOTAX™]: an intracellularly targeted taxane, Anti-Cancer Drug 16 (2005) 243–254. [6] Y. Matsumura, H. Maeda, A new concept for macromolecular therapies in cancer chemotherapy: mechanism of tumouritropic accumulation of proteins and the antitumour agent SMANCS, Cancer Res. 6 (1986) 6387–6392. [7] P.A. Vasey, S.B. Kaye, R. Morrison, C. Twelves, P. Wilson, R. Duncan, A.H. Thomson, L.S. Murray, T.E. Hilditch, T. Murray, S. Burtles, D. Fraier, E. Frigerio, J. Cassidy, Phase I clinical and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: first member of a new class of chemotherapeutic agents–drug–polymer conjugates, Clin. Cancer Res. 5 (1999) 83–94. [8] T. Minko, P. Kopeckova, V. Pozharov, J. Kopecek, HPMA copolymer bound adriamycin overcomes MDR1 gene encoded resistance in a human ovarian carcinoma cell line, J. Control. Release 54 (1998) 223–233. [9] R. Duncan, N-(2-Hydroxypropyl) methacrylamide copolymer conjugates, in: G.S. Kwon (Ed.), Polymeric Drug Delivery System, Marcel Dekker, Inc., New York, 2005, pp. 1–92. [10] R. Duncan, Polymer–drug conjugates, in: D. Budman, H. Calvert, E. Rowinsky (Eds.), Handbook of Anticancer Drug Development, Lippincott Williams and Wilkins, Philadelphia, 2003, pp. 239–260. [11] M.J. Vicent, S. Manzanaro, J.A. de la Fuente, C. Pérez, R. Duncan, HPMA copolymer–1,5-diazaanthraquinone conjugates as promising anticancer therapeutics, J. Drug Target. 12 (2004) 503–515. [12] R. Satchi-Fainaro, M. Puder, J.W. Davies, H.T. Tran, D.A. Sampson, A. Greene, G. Corfas, J. Folkman, Targeting angiogenesis with a conjugate of HPMA copolymer and TNP-470, Nat. Med. 10 (2004) 225–261.

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