CD44-Targeted Polymer–Paclitaxel Conjugates to ... - ACS Publications

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

CD44-Targeted Polymer−Paclitaxel Conjugates to Control the Spread and Growth of Metastatic Tumors Michal Zaiden,† Marie Rütter,† Lina Shpirt,† Yvonne Ventura,† Valeria Feinshtein,† and Ayelet David*,†,‡ Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, and ‡Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel

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ABSTRACT: One of the greatest challenges in cancer therapy is to control metastatic spread, seeding, and growth of tumors in distant organs. Recently, we reported on the design of a novel “drug-free” therapeutic copolymer bearing the antimigratory A5G27 peptide, designated P-(A5G27)-FITC, that shows excellent specificity to cancer cells overexpressing CD44v3 and CD44v6 and inhibits cancer cell migration and invasion. We demonstrated that P-(A5G27)-FITC accumulated preferentially in subcutaneous (sc) implanted 4T1 tumors following parenteral administration. Moreover, we showed that pretreatment of mice with P-(A5G27)-FITC prior to 4T1 cell inoculation inhibited colonization of circulating 4T1 cells in the lungs. In this study, we designed a new polymer-peptide-drug conjugate to inhibit vigorously growing primary tumors and control invasive behavior of cancer cells. To this end, the antimitotic drug (paclitaxel, PTX) was conjugated to P-(A5G27)-FITC. The targeted polymer−drug conjugate (P-(A5G27)-PTX) was significantly more toxic toward CD44overexpressing cancer cells than the nontargeted copolymer. In vivo, a single iv injection of P-(A5G27)-PTX prolonged the survival of C57BL/6 mice with established B16-F10 lung metastases. When injected intraperitoneally into BALB/c mice implanted sc with 4T1 tumors, P-(A5G27)-PTX significantly decreased the rate of primary tumor growth, increased the median survival of mice, and reduced the number of 4T1 metastases in the lungs when compared to nontargeted copolymer. Most interestingly, the CD44-targeted “drug-free” copolymer P-(A5G27) (without PTX) significantly inhibited the rate of tumor growth and further prolonged the median survival time of mice to the same extent as the PTX-containing formulations (P(A5G27)-PTX or free PTX). Overall, this study highlights the therapeutic potential of the HPMA copolymer−A5G27 conjugates (“drug-free” and PTX-bearing copolymers) to control the metastatic spread of cancer. KEYWORDS: CD44, invasion, metastasis, paclitaxel, polymer−drug conjugates, targeted cancer therapy

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INTRODUCTION Metastasis, the dissemination of cancer cells from the primary tumor to distant tissues, is the primary cause of cancer morbidity and mortality.1,2 CD44, a cell adhesion molecule and the primary receptor for hyaluronic acid (HA), is involved in many cell−cell interactions, cell adhesion, and cell migration. The standard isoform CD44s is normally expressed on the surface of most cell types. CD44 splice variants CD44v3 and CD44v6 are upregulated in many invasive cancer cells (including breast, colon, prostate, and lung cancer) and cancer stem cells and promotes metastasis by enhancing cell migration and invasion.3−6. Moreover, elevated CD44v3/6 level on circulating cancer cells is associated with increased distant recurrence and reduced disease-free survival in patients.7 We thus hypothesized that the interference with CD44v3/6mediated activity could inhibit the malignant phenotype, slow cancer progression, and decrease invasiveness. The laminin-α 5 chain-derived synthetic peptide A5G27 has been previously shown to bind specifically to the glycosami© XXXX American Chemical Society

noglycan (GAG) side chains of CD44v3 and CD44v6 and to inhibit cancer cell migration, invasion, and angiogenesis.8−10 In vivo, the peptide inhibited primary B16-F10 tumor growth, angiogenesis, lung colonization, and tumor progression. We have recently synthesized a new “drug-free” therapeutic copolymer, carrying multiple copies of the A5G27 peptide, to inhibit cancer cell migration and invasion.11 The watersoluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer−A5G27 conjugate (P-(A5G27)-FITC) showed remarkable specificity to CD44-overexpressing cancer cells and inhibited in vitro cancer cell migration and invasion. We further showed that pretreatment of mice with P-(A5G27)Special Issue: New Directions for Drug Delivery in Cancer Therapy Received: March 14, 2018 Revised: May 23, 2018 Accepted: June 5, 2018

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DOI: 10.1021/acs.molpharmaceut.8b00269 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 1. Scheme for the synthesis of the FITC-labeled, CD44-targeted HPMA copolymer−paclitaxel conjugate (P-(A5G27)-PTX).

This study is the first experiment testing whether the combination of A5G27 with a conventional chemotherapeutic drug on the same polymeric carrier could improve in cancer therapy.

FITC (by a single intraperitoneal (ip) injection) significantly inhibited colonization of circulating 4T1 cells in the lungs, demonstrating in vivo the antimigratory features of the new “drug-free” copolymer. Moreover, P-(A5G27)-FITC accumulated preferentially in subcutaneous 4T1 tumors owing to the enhanced permeability and retention (EPR) effect,12 thus suggesting its potential utility for cancer imaging and therapy. In this study, the P-(A5G27)-FITC copolymer was further conjugated with an antimitotic agent, paclitaxel (giving rise to P-(A5G27)-PTX), to inhibit primary tumor growth and invasive behavior of vigorously growing cancer cells. PTX is used for the treatment of metastatic breast and ovarian cancer, melanoma, non-small-cell lung carcinoma, and many other cancers.13 Because B16-F10 cells use CD44 as a primary receptor for disease progression and metastatic behavior in the lungs,14 the impact of P-(A5G27)-FITC and P-(A5G27)-PTX on the in vivo metastatic potential of B16-F10 melanoma cells was tested in an experimental lung metastasis model. The ability of P-(A5G27)-PTX to inhibit the formation of breast cancer metastasis was also tested in a murine 4T1 breast cancer model. When injected subcutaneously (sc) into syngeneic BALB/c mice, 4T1 cells form primary tumors that are able to complete all steps of the metastatic process (i.e., invasion, intravasation, transport, arrest, extravasation, and growth), resulting in a large number of metastatic lung nodules.15 P(A5G27)-PTX was administered ip to mice bearing sc inoculated 4T1 tumors, and the effect on tumor growth rate, mice survival, and lung metastasis formation was measured.

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MATERIALS AND METHODS Materials. N-Terminal N-acetylated lysine-harboring A5G27 peptide (K-A5G27, primary sequence Ac-KRLVSYNGIIFFLR) and the peptide with a scrambled A5G27 sequence A5G27scrm (K-A5G27scrm, Ac-KVLFGFLRIYSRIN) were purchased from GL Biochem (Shanghai, China). The C-terminal lysine in the original sequence9 was substituted by arginine to avoid reactions with reactive ester groups on the polymer precursor (see below). The catalyst 4-dimethyl aminopyridine (DMAP) was purchased from Novabiochem-Merck (Darmstadt, Germany). PTX was purchased from LC Laboratories (Woburn, MA). 4-Oxopentanoic acid (levulinic acid, Lev) was purchased from Acros Organics (Geel, Belgium). Methacryloyl-aminopropyl (MAP) was purchased from Polysciences (Warrington, PA). All other chemicals, reagents, and solvents were purchased from Sigma-Aldrich (Rehovot, Israel). HPMA,16 methacryloylglycylglycine p-nitrophenyl ester (MA-GG-ONp),17 methacryloyl-glycyglycine hydrazone-Boc (MA-GG-HZBoc),18 and methacryloyl-aminopropyl fluorescein-5-isothiocyanate (MAP−FITC)19 were synthesized as described previously. Cell Lines. 4T1 murine mammary carcinoma cells were purchased from the American Type Culture Collection B

DOI: 10.1021/acs.molpharmaceut.8b00269 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Table 1. Characteristics of the HPMA Copolymer−A5G27 Conjugates and Control Copolymers HPMA conjugate P-(GG-ONp)-FITC P-(GG-ONp)-(GGHZBoc)-FITC P-(A5G27) P-(A5G27scrm) P-(A5G27)-PTX P-(A5G27scrm)-PTX P-(A5G27)-PTX P-(A5G27scrm)-PTX P-(GG-OH)-PTX P-(A5G27)

polymer type precursor 1 for “drug-free” copolymers precursor 2 for polymer− drug conjugates targeted for in vitro binding nontargeted for in vitro binding targeted for MTT assay nontargeted for MTT assay targeted for in vivo studies nontargeted for in vivo studies nontargeted for in vivo studies targeted for MTT and in vivo studies

∼Mw (kDa)a

PIa

36.8

1.59

24.1

1.60

41.1 41.1

1.92 1.92

1.30 1.30

3 3

24.1 24.1 24.1 24.1

1.60 1.60 1.60 1.60

1.30 1.30 1.30 1.30

4.5 5.5 8.9 8.3

33.5

1.25

1.35

36.8

1.59

1.77

mol % ONpb 10 8.9

mol % FITCc

mol % BOCd

mol % peptidee

drug content, wt % (mol %)f

free drug (mol % of drug)f

1.23 (0.38) 1.22 (0.42) 2.3 (0.96) 1.75 (0.7)

0.014 0.021 0.02 0.06

1.77 1.30

8

7.8 (1.7)

0.23

5.7

a The apparent molecular weight (Mw) and polydispersity (PI) of copolymers were estimated by size-exclusion chromatography (SEC) on an AKTA FPLC system using a Sephacryl S-400 column (GE Healthcare) with calibrated polyHPMA fractions. bDetermined spectrophotometrically by measuring UV absorbance at 400 nm (ε = 17,700 M−1 cm−1). cDetermined by measuring UV absorbance at 492 nm (ε = 82,000 M−1 cm−1). d Determined by 1H NMR at 500 Hz in D2O using the Boc t-butyl protons chemical shift (δ 1.40, s, 9H) for calculation. eEstimated by 1H NMR at 500 Hz using the tyrosine (Y) and 2× phenylalanine (F) proton chemical shifts (δ 6.5−8, m, 14H). fDetermined using the HPLC system equipped with a reverse-phase column (Symmetry Silica C18, 5 μm, 4.6 × 250 mm column, acetonitrile−water gradient, UV−vis photodiode array detector, at 230 nm).

Synthesis of FITC-Labeled CD44-Targeted Copolymer P-(A5G27). K-A5G27 was bound to P-(GG-ONp)-FITC by aminolysis of reactive ONp groups, as described.20 Briefly, P(GG-ONp)-FITC (50 mg, 1.4 × 10−3 mmol) was dissolved in anhydrous DMSO (300 μL) containing triethylamine (50 μL) and added to K-A5G27 (30 mg, 1.7 × 10−2 mmol) dissolved in anhydrous DMSO (200 μL) and stirred for 48 h at 37 °C. The conjugate was incubated for 1 h in 1N NaOH solution to release unreacted ONp groups and purified on a PD-10 column. The characteristics of the FITC-labeled copolymer, henceforth referred to as P-(A5G27), are summarized in Table 1. The control copolymer with a scrambled A5G27 sequence (P-(A5G27scrm)) was synthesized and purified similarly. A control copolymer without any peptide (P-(GG-OH)-FITC) was obtained by aminolysis of the ONp groups from P-(GGONp)-FITC in 1 N NaOH solution. Synthesis of CD44-Targeted HPMA Copolymer−PTX Conjugate P-(A5G27)-PTX. A keto-carbonyl functional group was first introduced to PTX by reacting the 2′-OH of PTX with levulinic acid (LEV) according to the procedure previously described by Etrych et al.21 Briefly, LEV (19.37 mg, 0.166 mmol in 100 μL of DMF) and DCC (37.5 mg, 0.182 mmol in 200 μL of DMF) were mixed and cooled for 20 min to −18 °C. Then, a solution of PTX (100 mg, 0.117 mmol) and 4-dimethyl aminopyridine (DMAP, 14 mg, 0.117 mmol) in 300 μL of DMF was added. The reaction proceeded at 4 °C for 72 h. The precipitated DCU was filtered off, and the reaction mixture was purified on a silica gel 60 (1.6 × 30 cm) column using ethyl acetate as eluent. Eluent containing PTXLEV was collected and concentrated, and the pure compound was precipitated in diethyl ether and dessicated. The yield was 67.5 mg (56.5%). The final product was characterized using MALDI-TOF analysis. Calculated: 951 g/mol; found: 952, 974, and 990 g/mol corresponding to M + H+, M + Na+, and M + K+, respectively. The purity of PTX-LEV was determined by HPLC (waters 2695) equipped with a waters Symmetry C18 column (4.6 × 250 mm, 5 μm) with a UV−vis

(ATCC, Manassas, VA). A luciferase-expressing clone of 4T1 cells (4T1-Luc) were provided by Prof. Ron N. Apte (BenGurion University of the Negev, Israel). 4T1 and 4T1-Luc cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, 100 IU/ml penicillin, and 100 μg/mL of streptomycin (all from Biological Industries Ltd., Beit Haemek, Israel). Murine B16-F10 melanoma cells were purchased from ATCC. B16-F10 was cultured in DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 μg/mL of streptomycin The cells were maintained in an incubator at 37 °C in a humidified atmosphere with 5% CO2. Mice. Female BALB/c and female C57BL/6 mice were obtained from Envigo CRS (Ness-Ziona, Israel). All experimental procedures were approved and performed in compliance with the standards of Ben-Gurion University of the Negev Institutional Animal Care and Use Committee. Mice at 6−8 weeks of age were housed in accordance with approved institutional guidelines. Synthesis of Precursor Copolymers P-(GG-ONp)-FITC and P-(GG-ONp)-(GG-HZBoc)-FITC. The polymer precursor P-(GG-ONp)-FITC bearing O-nitrophenyl (ONp) ester groups for further peptide (A5G27 or A5G27scrm) attachment were prepared by radical precipitation copolymerization of HPMA, MA-GG-ONp, and MAP-FITC at 88:10:2 mol %, respectively, as described previously.20 The polymer precursor P-(GG-ONp)-(GG-HZBoc)-FITC bearing ONp and tertbutyloxycarbonyl (Boc)-protected hydrazone groups, which were later used for peptide and PTX attachment, respectively, was synthesized similarly by copolymerizing HPMA, MA-GGONp, MA-GG-HZBoc, and MAP-FITC at 80:8:10:2 mol %, respectively (Figure 1).20 The polymers were precipitated into cold diethyl ether, desiccated, and purified on an LH-20 column (1.6 × 60 cm) using methanol as eluent. The characteristics of the synthesized copolymers are summarized in Table 1. C

DOI: 10.1021/acs.molpharmaceut.8b00269 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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were collected after trypsinization of cell monolayers. Cells (5 × 105) were incubated in 3% BSA for 30 min at room temperature and then treated with 50 μg/mL of the FITClabeled P-(A5G27) or P-(A5G27scrm) in 0.5 mL of PBS containing 1% BSA for 1 h at 4 °C or for 4 h at 37 °C. Cells were then washed twice with PBS, and the fluorescence intensity was analyzed by Guava easyCyte flow cytometer (EMD Millipore, excitation at 488 nm and emission at 525 nm). Cytotoxicity Determination. 4T1 (750 cells/well) cells were seeded on 96-well plates with completed RPMI 1640 medium and incubated for 24 h (37 °C, 5% CO2). The cells were then exposed to PTX, P-(A5G27)-PTX, P-(A5G27scrm)PTX, and P-(A5G27) in serial dilutions at an equivalent dose of PTX or A5G27 for 72 h. Cell viability was determined by MTT assay as described.23 Inhibiting the Growth of Established B16-F10 Melanoma Lung Metastases. Wild-type C57BL/6 mice (8 weeks old) were injected via the tail vein with 1 × 105 B16F10 cells on day 0. On day 7 after tumor cell inoculation, the mice were randomly divided into four groups of four mice and treated by a single iv injection of free PTX at a dose of 15 mg/ kg formulated in Cremophor EL vehicle (1:1:8 Cremophor EL:ethanol:PBS), P-(A5G27)-PTX, or P-(GG-OH)-PTX at a dose of 15 mg/kg of PTX equivalent. The control group was left nontreated (only PBS). The body weights of the mice and survival were monitored (Kaplan−Meier method). Inhibiting 4T1 Breast Cancer Tumor Growth and the Formation of Lung Metastases. 4T1-Luc cells (2 × 105/ mouse) were inoculated sc in the fourth mammary fat pad area of female BALB/c mice (8 weeks old). Three days later, tumors were visible and palpable. Treatment started on the fourth day after 4T1-Luc cell inoculation with the PTX dose of 15 mg/kg, totaling 2 ip injections at days 4 and 10 postinoculation. Experimental groups included free PTX formulated in Cremophor EL vehicle (1:1:8 Cremophor EL:ethanol:PBS), P-(A5G27)-PTX, P-(A5G27scrm)-PTX (at a 15 mg/kg PTX equivalent dose), P-(A5G27) (equivalent peptide dose relative to P-(A5G27)-PTX), and nontreated control (saline). Mice body weight, tumor volume, and survival were monitored (Kaplan−Meier method). Lung metastasis was monitored from day 21 by ip injection of 150 mg/kg of Dluciferin (Promega, WI, USA), and luminescence was measured with an in vivo imaging system (IVIS Lumina Imaging System; PerkinElmer) 15 min after the D-luciferin injection. Statistical Analysis. A one-way ANOVA test followed by Bonferroni corrections was used to determine statistically significant differences between experimental and control groups. The Log-rank (Mantel-Cox) and Gehan-BreslowWilcoxon tests were used to determine differences in the survival time between experimental and control groups. Differences were considered significant if the p-value was less than 0.05. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001.

photodiode array detector (waters 996). HPLC analysis was performed with an eluent of water−acetonitrile with a gradient of 10−90 vol % acetonitrile in water, showing a peak at 21.4 min (λ = 230 nm). HPMA copolymer−PTX conjugates with A5G27 or A5G27scrm were prepared based on our previously described procedure.22 Briefly, K-A5G27 was first conjugated to P-(GGONp)-(GG-HZBoc)-FITC precursor copolymer via ONp aminolysis followed by removal of Boc groups from the protected copolymers by concentrated TFA for 5 min. The resulting P-(A5G27)-(GG-NHNH2)-FITC (50 mg, 2 × 10−3 mmol) was dissolved in anhydrous methanol (10 wt % solution), and PTX-LEV (15 mg, 1.57 × 10−2 mmol, 60 mol % relative to the hydrazide groups content) was then added under stirring in the dark. The reaction mixture was stirred for 48 h after addition of catalytic amounts of acetic acid (50 μL/ ml of reaction mixture) and then purified on a Sephadex LH20 column (1.6 × 60 cm) using methanol as eluent.22 The polymer fractions henceforth referred to as P-(A5G27)-PTX were collected and concentrated, and the polymer conjugate was precipitated into cold diethyl ether. The total drug content was determined by HPLC after complete hydrolysis of a known amount of the copolymer in 1 mL of HCl solution (pH 2) for 24 h at room temperature followed by extraction of the released PTX-LEV into chloroform. In addition, the content of free PTX-LEV was determined by HPLC after extraction of the respective drug from an aqueous solution of the conjugate to chloroform (Table 1). The control polymer−PTX conjugate with a scrambled A5G27 sequence (P-(A5G27scrm)-PTX) was synthesized similarly. P-(GG−OH)-PTX (without a peptide) was prepared by attaching PTX to the P-(GGONp)-(GG-HZBoc)-FITC precursor from which the ONp groups were first released in 1 N sodium hydroxide solution. Determination of CD44 Expression Levels on Cancer Cells. Suspensions of 4T1-Luc and B16-F10 cells (5 × 105) were collected and incubated in 3% BSA for 30 min at room temperature to block nonspecific binding sites. The cells were then incubated for 1 h at 4 °C with rat anti-mouse CD44 antibodies (clone 7, BioLegend, CA, USA, 1:50 dilution) followed by incubation with AlexaFluor 488-conjugated antirat secondary antibodies (Jackson ImmunoResearch Laboratories Inc., PA, USA, 1:100 dilution) for an additional 30 min. Cellassociated fluorescence was determined by flow cytometry using a Guava easyCyte Flow Cytometer (excitation at 488 nm, emission at 525 nm). In Vitro Release of PTX from Polymer−PTX Conjugates. The rate of PTX release, free and/or ester derivatives, from P-(A5G27)-PTX was determined by incubating P(A5G27)-PTX in phosphate buffers at pH 5.0 or 7.4 (0.1 M phosphate buffer with 0.05 M NaCl) at 37 °C, as described previously.21 The concentration of the conjugate in solution was equivalent to 0.01 mM PTX. The amount of released drug was determined by HPLC analysis after extraction of the released PTX or PTX-LEV into chloroform. Analysis was performed on an HPLC at 230 nm, as described above. HPLC analysis, using a water−acetonitrile gradient of 10−90% acetonitrile, showed a peak at 20.0 and 21.4 min for PTX and PTX-LEV, respectively. The percentage of released drug is expressed as the amount of free drug relative to the total drug content in the conjugates. All experiments were carried out in duplicate. Binding and Uptake of CD44-Targeted Copolymer by CD44-Expressing Cancer Cells. 4T1-Luc and B16-F10 cells

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RESULTS Synthesis and Characterization of Copolymers. Two FITC-labeled HPMA-based precursor copolymers were first synthesized in this study by radical polymerization: precursor 1 (P-(GG-ONp)-FITC) for the synthesis of “drug-free” polymer-peptide conjugates and precursor 2 (P-(GG-ONp)(GG-HZBoc)-FITC) for further peptide and PTX attachment. D

DOI: 10.1021/acs.molpharmaceut.8b00269 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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it to that obtained for the same experiments with free PTX, as described previously.22 The free PTX exhibited the lowest IC50 dose owing to the rapid diffusion into the cells (Table 2). P(A5G27)-PTX was ∼4-fold more toxic than the nontargeted P(A5G27scrm)-PTX copolymer, suggesting a faster (receptormediated) internalization (Figure 4). In addition, cells incubated with very high concentrations of drug-free copolymer (P-(A5G27)) for 72 h were not affected by the peptide (at a range of 0.01−100 μM). P-(A5G27)-PTX Inhibits the Growth of Established B16-F10 Lung Metastases. PTX has been shown to enhance the survival rate of B16-F10 melanoma tumor-bearing mice.13 A5G27 peptide was reported to inhibit primary B16-F10 tumor growth, invasion, angiogenesis, and in vivo lung colonization.8 In the present study, we tested whether the combination of PTX with A5G27 peptide on the same polymeric backbone (P-(A5G27)-PTX) would inhibit the growth of established B16-F10 pulmonary metastases and thereby prolong the survival time of mice compared to that of free PTX. C57BL/6 mice were injected via the tail vein with B16-F10 cells on day 0 and treated iv with polymers (P(A5G27)-PTX, P-(GG-OH)-PTX) or with free PTX 7 days post tumor cell inoculation. P-(A5G27)-PTX prolonged mice survival in 7 and 20% relative to the survival rates of free PTX and nontreated mice, respectively (Figure 5). The results also indicate that both polymer−PTX conjugates (P-(A5G27)-PTX and P-(GG-OH)-PTX) slightly improved the therapeutic effect relative to free PTX. Differences between treatment groups were not significant. P-(A5G27)-PTX Inhibits the Rate of Tumor Growth, Increases Median Survival of Mice, and Decreases the Number of 4T1-Luc Pulmonary Metastases. Next we investigated whether polymer−A5G27 conjugates (with or without PTX) can inhibit the formation of breast cancer lung metastases in a mouse orthotopic tumor model in which luciferase-labeled 4T1 mammary tumor cells metastasized to the lung region after 21−28 days in BALB/c mice. Two sequential ip injections of P-(A5G27)-PTX (at 15 mg/kg PTX equivalent dose) statistically significantly inhibited the rate of tumor growth (Figure 6A) (p < 0.001; from day 32) when compared to P-(A5G27scrm)-PTX, free PTX, or nontreated mice. Interestingly, the “drug-free” copolymer, P-(A5G27) (at an equivalent A5G27 dose relative to P-(A5G27)-PTX), also significantly inhibited tumor growth (p < 0.001) when compared to P-(A5G27scrm)-PTX, free PTX, or nontreated mice. P-(A5G27)-PTX significantly prolonged the survival time of tumor-bearing mice when compared to nontreated mice (p < 0.05). The median survival of mice in the P(A5G27)-PTX treatment group was longer than in P(A5G27scrm)-PTX, P-(A5G27), and free PTX-treated groups (48.50 vs 40.5, 43, and 45.5, respectively); however, differences were nonsignificant (Figure 6B). The median survival time of the P-(A5G27)-treated group was slightly higher than the P(A5G27scrm)-PTX-treated and nontreated mice and was not statistically significant (43 vs 40.5 and 39, respectively). Body weight measurements showed no reduction in mice weight in polymer-treated groups (Figure 6C), suggesting that HPMA copolymer−A5G27 conjugates did not cause toxicity in vivo. Moreover, the profile of optical imaging from selected mice in different experimental groups showed visible lung metastases in the nontreated group from day 28 (n = 2 out of 4 surviving mice) and in free PTX, P-(A5G27scrm)-PTX, and P(A5G27)-PTX treated mice only from day 32 (n = 2 out of

Second, A5G27 or A5G27scrm were coupled to the precursor copolymers via ONp aminolysis. The keto-carbonyl-modified PTX was then attached to the hydrazide group-terminated side chains of the polymer (P-(A5G27)-(GG-NHNH2)-FITC to facilitate pH-controlled chemical hydrolysis at mildly acidic pH.21 The average molecular weight of the resulting precursor 1 and 2 copolymers was determined to be ∼37 and 24 kDa, respectively, with a dispersity of 1.6. Spectrophotometric analysis showed that P-(A5G27) contained ∼3 mol % of peptide. The content of the peptide was higher in the CD44targeted polymer−PTX conjugates (5.7−8.9 mol %). PTX content in polymer−peptide conjugates was relatively low, 1.2−2.3 wt %, and higher (7.8 wt %) in the peptide-free copolymer (P-(GG-OH)-PTX). Increasing the molar ratio between free PTX to hydrazide groups on P-(A5G27)-(GGNHNH2)-FITC or prolongation of the reaction time up to 5 days did not further increase the drug content in the copolymer. The polymer−drug conjugates contained less than 0.2 mol % of free PTX derivatives. P-(A5G27)-PTX was stable in vitro under physiological pH (7.4) with ∼30% of PTX and PTX-LEV released after 24 h (Figure 2). Drug (PTX

Figure 2. Analysis of PTX and PTX-LEV release from P-(A5G27)PTX, as determined by HPLC. The polymer-bound PTX was incubated in phosphate buffers at pH 5 and pH 7.4 at 37 °C. n = 2.

and PTX-LEV) release from P-(A5G27)-PTX was faster at pH 5 than at pH 7.4. As expected, PTX release was faster at pH 5 with ∼50% of released PTX determined after 1 h of incubation and almost 90% of drug released after 24 h. P-(A5G27) binds Preferentially to CD44-Overexpressing Cancer Cells. CD44 expression levels in 4T1-Luc and B16-F10 cells were first determined using flow cytometry analysis. 4T1-Luc and B16-F10 cells expressed high levels of CD44 (Figure 3A).24,25 The cellular uptake of the CD44targeted conjugates was then evaluated. P-(A5G27) was more significantly bound (after 1 h, Figure 3B) (p < 0.001) and internalized (after 4 h, Figure 3C) by CD44-overexpressing cells than P-(A5G27scrm) (p < 0.001 in 4T1-Luc and p < 0.01 in B16-F10 cells). Cells treated with the control copolymer with a scrambled A5G27 peptide (P-(A5G27scrm) exhibited a higher percentage of labeled cells compared to nontreated cells. P-(A5G27)-PTX Is Highly Toxic toward Cells Expressing High CD44 Levels. The cytotoxicity of P-(A5G27)-PTX was studied by following its growth inhibition activity against 4T1 cells after 72 h exposure to the copolymer and comparing E

DOI: 10.1021/acs.molpharmaceut.8b00269 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 3. P-(A5G27) binds preferentially to CD44-overexpressing cells. (A) Flow cytometry analysis of CD44 expression in 4T1-Luc and B16-F10 cells. (B) Binding (1 h at 4 °C) and (C) internalization (4 h at 37 °C) of P-(A5G27) and P-(A5G27scrm) to 4T1-Luc and B16-F10 cells as determined by flow cytometry analysis. **p < 0.01, ***p < 0.001.

Table 2. IC50 Values (in μM) for Inhibition of Proliferation of 4T1 Cells Following Treatment with Free or PolymerBound PTX drugs

IC50 (in μM)

PTX P-(A5G27)-PTX P-(A5G27scrm)-PTX P-(A5G27)

0.026 ± 0.006 0.058 ± 0.015 0.221 ± 0.055 NDa

a

ND = non-detectable.

Figure 5. Kaplan−Meier curve for survival of mice bearing B16-F10 lung metastases. Mice were injected iv with free PTX (15 mg/kg) and P-(A5G27)-PTX or P-(GG-OH)-PTX (at 15 mg/kg PTX equivalent dose) 7 days post B16-F10 inoculation and were monitored for survival for 55 days. n = 5 for each treatment group.

targeted polymer−peptide conjugates20 and polymer−cell penetrating peptide (CPP) conjugates27 have been applied by our group to improve drug delivery and penetration to lung metastases and led to a significant increase in the survival rate of mice in the experimental B16-F10 lung metastasis model. Very recently, we demonstrated that the “drug-free” copolymer P-(A5G27) binds specifically to CD44-overexpressing cells, and inhibit cell migration and invasion.11 Moreover, we found that pretreatment of mice with P-(A5G27) inhibits metastatic seeding of circulating 4T1 cells in the lungs owing to the antimigratory properties of A5G27 peptide. In the present study, we tested whether the combination of A5G27 and PTX on the same HPMA polymeric backbone would produce improved therapeutic efficacy in highly metastatic tumor models. B16-F10 cells with high lung colonization capacity28 were selected to evaluate the antimetastatic activity of the polymers in an established B16-F10 lung metastasis model. We also tested the activity of the polymers against sc 4T1mammary tumors that share many characteristics with human

Figure 4. Cytotoxic activity of HPMA copolymer−A5G27 conjugates. 4T1 cells were incubated with free PTX and HPMA copolymer−PTX conjugates for 72 h.

6 surviving mice) (Figure 7). P-(A5G27)-PTX, P-(A5G27), and free PTX-treated mice showed decreased numbers of lung metastases on day 37 in comparison to P-(A5G27scrm)-PTX and nontreated mice (n = 3 out of surviving mice).

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DISCUSSION Systemic delivery of drugs to primary and metastatic tumors can be significantly improved by utilizing polymer−drug conjugates that extend circulation time and improve the overall drug accumulation in tumors.26 Furthermore, actively F

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Figure 6. Anti-tumor efficacy of polymer−A5G27 conjugates in 4T1 tumor-bearing mice. (A) Tumor growth was measured after treatment with “drug-free” copolymer P-(A5G27) and PTX-containing formulations (P-(A5G27)-PTX, P-(A5G27scrm)-PTX, or free PTX, 15 mg/kg PTX equivalent dose) in two ip injections on days 4 and 10. (B) Survival curves for 4T1-Luc tumor-bearing mice treated with polymer−A5G27 conjugates either free or PTX conjugated. The survival rates are presented as Kaplan−Meier curves. The survival curves of individual groups were compared by a log rank test. (C) Relative changes in body weight over the duration of the experiment. Tumor growth and body weights were monitored until day 37.

Figure 7. P-(A5G27) and P-(A5G27)-PTX treatment inhibited the lung metastasis of 4T1-Luc tumors. Luminescence of metastatic lung tumors was measured in selected mice by IVIS on day 28 (n = 2), 32 (n = 2), and 37 (n = 3) post 4T1-Luc cell inoculation. For facilitating metastases detection in the lungs, the lower portion of each animal was shielded with black paper to block luminescence from the primary tumor.

improve pharmacokinetics and tumor delivery of PTX.21 Recently, we showed that the HPMA copolymer−A5G27 conjugate (without PTX) at a hydrodynamic radius of ∼4 nm and Mw < 40,000 kDa improves polymer accumulation at the

breast cancer, particularly its ability to metastasize to the lungs, bones, and lymph nodes.29 PTX conjugation to HPMA copolymer chain (without A5G27) has been previously shown to increase drug solubility, G

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“drug-free” macromolecular therapeutics (polymer without conventional chemotherapy) for inhibiting cancer progression and metastasis. The paradigm of “drug-free” macromolecular therapeutics, first proposed by Kopecek and colleagues, constitutes the design of new polymer-based nanomedicines that lack low-MW cytotoxic compounds and thus the absence of nonspecific toxicities. The “drug-free” macromolecular therapeutics are typically grafted with multiple copies of targeting moieties on a flexible polymeric chain to facilitate multivalent interactions with cell surface receptors, resulting in receptor clustering (cross-linking) and apoptosis.30−32 The new “drug-free” macromolecular therapeutics presented herein effectively target CD44-overexpressing cancer cells, inhibit cancer cell migration and invasion,11 and suppress the growth of vigorously growing 4T1 primary tumors. The demonstrated ability of A5G27 solely to inhibit primary tumor growth and lung colonization of cancer cells9,11 can explain the significant therapeutic activity of P-(A5G27) copolymer. The contribution of CD44 receptor clustering-mediated apoptosis will be tested in future experiments to learn about the mechanism of the in vivo activity of P-(A5G27). Overall, our data suggest a new, CD44-targeted, clinically relevant therapy for inhibiting the development and progression of cancer. Neither P-(A5G27) nor P-(A5G27)-PTX induced apparent toxicity at the tested dose, suggesting the safety of our polymers. P-(A5G27)-PTX demonstrated higher but comparable therapeutic efficacy relative to P-(A5G27), and we failed to demonstrate a synergistic or additive antitumor effect as a result of the combination of antimigratory and antimitotic agents on the same polymeric backbone. The relatively low PTX loading in polymer conjugates can explain the lack of significant difference in the activity of P-(A5G27)PTX relative to P-(A5G27). It is possible that the low PTX loading was a result of the conjugation technique used in this experimental setting. The above-mentioned limitation can be overcome in future experiments by copolymerizing PTXcontaining monomer with HPMA and peptide-containing comonomers rather than using a precursor copolymer for PTX attachment. However, high loading with hydrophobic drugs can reduce both polymer solubility and the rate of drug release, which should be taken into account. Another explanation for the lack of synergy is that we treated the mice with only a single iv injection (in the B16-F10 model) or up to two ip injections (4T1-Luc sc tumors). We believe that a more aggressive dosing schedule (repetitive dosing) would improve P-(A5G27)-PTX activity and thus results in enhanced therapeutic outcomes. In conclusion, our current preclinical studies demonstrated that P-(A5G27)-PTX and P-(A5G27) decrease the rate of primary tumor growth, inhibit spreading of tumors in distant organs, and improve the median survival of P-(A5G27)-PTXtreated mice relative to nontargeted copolymer. We therefore suggest the A5G27-bearing copolymers as good candidates for preventing metastatic spreading of CD44-overexpressing cancer cells.

sc 4T1 tumors.11 We thus assumed that the combination of A5G27 and PTX on a single polymeric chain would enhance the antitumor and antimetastatic effects when compared to those of free drug. All the HPMA copolymer conjugates were grafted with multiple copies of peptide (A5G27 or A5G27scrm), and the drug (PTX) was attached to the polymeric backbone via a linker susceptible for hydrolytic degradation in mild acidic environment (in the case of drug-containing copolymers). The resulting 24−41 kDa copolymers presented a high peptide content and a relatively low drug content. The presence of oligopeptide led to smaller amounts of PTX in the conjugates, which was almost 4-times lower than in the control P-(GGOH)-PTX system, suggesting that the presence of A5G27 may have hindered PTX attachment. The rate of drug release from P-(A5G27)-PTX at pH 5 was much higher than that at pH 7.4. These properties should facilitate drug release in the slightly acidic tumor microenvironment and at intracellular compartments of cancer cells We first confirmed that the HPMA copolymer−A5G27 conjugate binds significantly to 4T1 and B16-F10 cells and then tested the cytotoxicity of the polymer−PTX conjugates toward 4T1 cells. The targeted copolymer, P-(A5G27), was bound and internalized more rapidly by CD44-overexpressing cancer cells than was the nontargeted control copolymer. Accordingly, the CD44-targeted polymer−drug conjugate (P(A5G27)-PTX) exhibited a 4-times higher cytotoxicity toward cells with high CD44 levels than P-(A5G27scrm)-PTX, suggesting a more rapid (CD44-mediated) internalization. Inhibiting the tumor growth of established tumor metastases in the lungs poses a significant challenge in cancer therapy. In the first preclinical experiments, we tested the ability of P(A5G27)-PTX to inhibit the growth of established lung metastases in mice induced by circulating B16-F10 melanoma cells. Mice injected with B16-F10 cells and treated with P(A5G27)-PTX (a single injection on day 7 after tumor inoculation) exhibited a prolonged survival time when compared to untreated mice; however, P-(A5G27)-PTX did not offer preclinical meaningful benefit over the passively targeted-P-(GG-OH)-PTX copolymer (at the same PTX equivalent dose) for the treatment of established B16-F10 lung metastases. For the ability of P-(A5G27)-PTX to inhibit spreading of vigorously growing tumors to distant organs to be tested, its therapeutic activity was evaluated in a second experimental setting of sc inoculated 4T1 tumors. Our results show that P-(A5G27)-PTX significantly inhibits the rate of tumor growth and led to prolonged median survival over P(A5G27), P-(A5G27scrm)-PTX, or free PTX treatment groups after only two sequential ip injections. This result supports that the combination of A5G27 and PTX on the same polymeric backbone showed superior antitumor effects than P-(A5G27) or P-(GGOH)-PTX of free PTX. P-(A5G27)-PTX offered higher median survival results than the free drug, probably due to prolonged circulation time and increased tumor accumulation; however, the difference was not statistically significant. The dosing regimen of P-(A5G27)-PTX could be further optimized in the future to improve the therapeutic efficacy. Interestingly, the CD44-targeted “drug-free” copolymer, P(A5G27) (without PTX), significantly inhibited the rate of tumor growth and decreased tumor progression and 4T1 lung colonization to the same extent as the PTX-containing formulations (polymer-bound PTX or free PTX). These results highlight once again the therapeutic potential of the

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AUTHOR INFORMATION

Corresponding Author

*Tel: +972-8-6477364; fax: +972-8-6479303; e-mail: [email protected]. ORCID

Ayelet David: 0000-0002-8929-5700 H

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants from the Israel Science Foundation (603/16) and the Israeli National Nanotechnology Initiative for a Focal Technology Area (FTA) on Nanomedicines for Personalized Theranostics.

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ABBREVIATIONS AIBN, 2,2′azobis(isobutyronitrile); DMAP, 4-dimethyl aminopyridine; DCU, dicyclohexylurea; EPR, enhanced permeability and retention; FITC, fluorescein-5-isothiocyanate; FPLC, fast protein liquid chromatography; GAG, glycosaminoglycan; GG, glycylglycine; HPMA, N-(2-hydroxypropyl)methacrylamide; LEV, levulinic acid; HA, hyaluronan, hyaluronic acid; HPLC, high-performance liquid chromatography; MA-FITC, 5-[3(methacryloylaminopropyl)thioureidyl]fluorescein; MA-GGOH, methacryloyl-glycylglycine; MA-GG-ONp, methacryloylglycylglycine p-nitrophenyl ester; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight; MS, mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolinium bromide; Mw, molecular weight; ONp, pnitrophenyl; PTX, paclitaxel; SEC, size-exclusion chromatography; TEA, triethylamine

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