pH Responsive Doxorubicin Delivery by Fluorous Polymers for Cancer

Jan 30, 2018 - Polymer–drug conjugates have played a crucial role in advancing pre-existing cancer chemotherapies by allowing for enhanced circulati...
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Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

pH Responsive Doxorubicin Delivery by Fluorous Polymers for Cancer Treatment Jaqueline D. Wallat, Jada K. Harrison, and Jonathan K. Pokorski* Department of Macromolecular Science and Engineering, Case Western Reserve University, Case School of Engineering, Cleveland, Ohio 44106, United States S Supporting Information *

ABSTRACT: Polymeric nanoparticles have emerged as valuable drug delivery vehicles as they improve solubility of hydrophobic drugs, enhance circulation lifetime, and can improve the biodistribution profile of small-molecule therapeutics. These nanoparticles can take on a host of polymer architectures including polymersomes, hyperbranched nanoparticles, and dendrimers. We have recently reported that simple low molecular weight fluorous copolymers can self-assemble into nanoparticles and show exceptional passive targeting into multiple tumor models. Given the favorable biodistribution of these particles, we sought to develop systems that enable selective delivery in acidic environments, such as the tumor microenvironment or the lysosomal compartment. In this report, we describe the synthesis and in vitro biological studies of a pH-responsive doxorubicin (DOX) fluorous polymer conjugate. A propargyl DOX hydrazone was synthesized and covalently attached to a water-dispersible fluorous polymer composed of trifluoroethyl methacrylate (TFEMA) and oligo(ethylene glycol) methyl ether methacrylate (OEGMEMA) using the ligand-accelerated copper-catalyzed azide−alkyne cycloaddition. Driven by the high fluorine content of the copolymer carrier, the DOX−copolymer formed stable micelles under aqueous conditions with a hydrodynamic diameter of 250 nm. The DOX−copolymer showed internalization into multiple in vitro models for breast and ovarian cancer. Cytotoxicity assays demonstrated efficacy in both breast and ovarian cancer with overall efficacy being highly dependent on the cell line chosen. Taken together, these results present a platform for the pH-triggered delivery of DOX from a fluorous micelle carrier effective against multiple cancer models in vitro. KEYWORDS: polymers, drug delivery, cancer chemotherapy

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INTRODUCTION Polymer−drug conjugates have played a crucial role in advancing pre-existing cancer chemotherapies by allowing for enhanced circulation lifetime, improved solubility of hydrophobic drugs, and enhanced biodistribution profiles over the unconjugated therapeutic.1,2 These functions are critical for reducing side effects and overcoming dose-limiting toxicity for standard chemotherapeutic treatment regimes. Polymer−drug conjugates come in many forms, but typically rely on watersoluble polymers to convey the aforementioned benefits.2 Due to the heterogeneity of tumor tissue and ability of tumors to develop drug resistance, it is critical to continue to develop new polymer−drug conjugates that can overcome dose-limiting toxicity and thus fight the development of drug resistance by fully eliminating tumor cells. As such, several new conjugates © XXXX American Chemical Society

have advanced to clinical trials and are a rapidly expanding research field.3−5 One therapy that has been used for decades is doxorubicin (DOX), an anthracycline that inhibits topoisomerase II. While this therapy has seen pervasive use in the clinic, there is a broad subset of cancers where it remains ineffective and off-target side effects are prevalent, providing great impetus to improve upon its use through polymer conjugation. Doxil is a nanoparticle drug delivery system composed of a PEGylated liposomal outer shell encapsulating DOX.6 Doxil Special Issue: Click Chemistry for Medicine and Biology Received: November 21, 2017 Revised: January 2, 2018 Accepted: January 16, 2018

A

DOI: 10.1021/acs.molpharmaceut.7b01046 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Scheme 1. Synthesis of 19F-DOX 3 via the Ligand Accelerated, Copper-Catalyzed Azide Alkyne Cycloaddition of DOX−Alkyne 1 and Fluorous Random Copolymer 2

effectively enhances solubility and plasma retention of DOX.7 DOX is a potent chemotherapeutic that is widely used in the treatment of various cancers and is limited by low solubility and interactions with off-target tissues, leading to severe negative side effects such as acute cardiotoxicity.8 Doxil improves the biodistribution, solubility, and toxicity profile of DOX, however extended plasma half-life increases the potential of long-term side effects, which has led to the clinical practice of doublefiltration plasmapheresis to eliminate Doxil from circulation.9,10 Furthermore, Doxil only shows limited efficacy for certain cancers, including Pt-resistant ovarian and triple negative breast cancers. Thus, there is an imperative to create DOX carriers that provide the ability to overcome dose-limiting toxicity of DOX and Doxil. In an effort to improve delivery and minimize systemic distribution of chemotherapeutics, a bevy of cancer-targeted drug carriers have been designed and investigated.11 Nanoparticle prodrugs have been developed to release their cargo at specific physiological pH.12−15 These nanoparticles respond to the relatively acidic endosomal and/or lysosomal compartments (pH ∼ 4.5−5.5) or the tumor microenvironment (pH ∼ 6.5), using degradable functional groups like acetals,16 esters,17 imines,18 or hydrazones19−22 to serve the purpose of pHtriggered delivery. Various nanocarrier architectures have been used to deliver DOX,23 including inorganic molecules,24−26 viruses,27−30 dendrimers,31,32 and polymeric structures.33−36 Polymeric nanoparticles are ideal candidates for drug delivery as they can have diverse and unique architectures such as hyperbranched particles, polymersomes, and micelles. These particles have therapeutic advantages of solubilizing lipophilic drugs, enhancing circulation lifetime, enabling high drug loading, and limiting off-target effects.37−39 A promising class of materials that remains underutilized for drug delivery is fluorinated polymers. Fluorinated materials have emerged in imaging applications as contrast agents, with extensive development for use in 19F MRI, as well as 18F PET.40,41 However, only few examples of fluorinated materials have been reported for drug delivery vehicles.42 In drug delivery, linear and star fluorous copolymers were electrostatically loaded with DOX and were effective in vitro against various breast cancer cells.43 Additionally, fluorinated polymeric materials have seen success in delivery of genetic material, improving transfection efficiency while limiting cellular cytotoxicity.44,45 We have recently described a low molecular weight telechelic copolymer of oligo(ethylene glycol) methacrylate (OEGMEMA) and trifluoroethyl methacrylate (TFEMA) that shows an excellent biodistribution profile in multiple tumor models.46 Furthermore, this polymer was able to both deliver a photosensitizing agent to in vitro skin cancer

models and enhance the ability to generate reactive oxygen species under hypoxic conditions.47 Given this promising data, we chose to utilize this platform to deliver more traditional chemotherapeutics in a pH responsive matter. Herein, we describe the use of a fluorous polymeric micelle that is covalently conjugated to DOX via a hydrazone linkage for pH responsiveness. The pH sensitive DOX−alkyne was fabricated using a hydrazone linkage and attached to a fluorous copolymer via the CuAAc reaction yielding a micelle conjugate with pH-selective drug release (19F-DOX, Scheme 1). Since the pH of tumors and of intracellular compartments is lower than physiological pH, the acid sensitive drug carrier is expected to show selective deposition of the drug at the tumor site or within tumor cells and mitigate dose-limiting toxicity. In previous studies with this copolymer carrier, passive tumor targeting showed exceptional biodistribution profiles for triple negative breast and ovarian cancer, tumors with no validated molecular markers. Furthermore, both of these tumor types are treated with DOX clinically and often with limited efficacy. Therefore, we aimed to evaluate the efficacy of 19F-DOX in a variety of cell lines associated with these devastating diseases. In order to test these hypotheses, the DOX−copolymer was synthesized, analyzed, and assayed in vitro against three ovarian cancer cell lines and a triple negative breast cancer model.

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EXPERIMENTAL SECTION Materials. Anhydrous dichloromethane, copper(I) bromide (98%), copper(II) bromide (99%+), trifluoroacetic acid, and deuterated chloroform (CDCl3) and methanol (CD3OD) were purchased from Acros Organic. tert-Butyl carbazate was purchased from Alfa Aesar. Fetal bovine serum was purchased from Atlanta Biologicals. Acetonitrile, chloroform (reagent grade), hydrochloric acid, and methanol (reagent grade) were purchased from Fisher Scientific. 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s modified Eagle medium (DMEM), penicillin−streptomycin, 0.05% (w/ v) trypsin−EDTA solution, and Roswell Park Memorial Institute (RPMI-1640) medium were purchased from Life Technologies. Bromoisobutyryl bromide (98%), 2-[2-(2chloroethoxy)ethoxy]ethanol (97%), N,N′-dicyclohexylcarbodiimide, 4-(dimethylamino)pyridine, 1,4-dioxane, oligo(ethylene glycol) methyl ether methacrylate, Mn ∼ 475 g/ mol (OEGMEMA), and 4-pentynoic acid were purchased from Sigma-Aldrich. Doxorubicin hydrochloride was purchased from TSZ Chemical. OEGMEMA and TFEMA were passed through basic alumina columns to remove the inhibitor prior to use. Cu(I)Br was purified by dissolving the crude material in glacial acetic acid, rinsing the remaining solid with ethanol, followed by diethyl ether, and drying in vacuo. 2,2-Bipyridine was B

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In Vitro Release Study. In order to assess release of the drug from the copolymer carrier, the copolymer conjugate (4 mg, n = 3) was dissolved in 500 μL of either phosphate buffer saline at pH = 7.4 or sodium acetate buffer, pH = 5.0, and the solutions were transferred into 3500 MWCO dialysis tubing. The dialysis tubing was then placed into a beaker containing 100 mL of the same buffer, and samples were incubated in an incubator and orbital shaker at 37 °C with horizontal agitation (90 rpm). To monitor release, 10 mL of buffer was removed and replaced with 10 mL of fresh buffer at time points of 1, 2, 3, 4, 12, 16, 24, 36, and 48 h. The total amount of released DOX was determined by monitoring absorbance of the resulting samples at 481 nm and applying the Beer−Lambert law to determine the concentration of DOX present to calculate a % cumulative release. Cell Culture. A2780 epithelial ovarian carcinoma cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin−streptomycin. OVCAR3 ovarian adenocarcinoma cells were maintained in RPMI-1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin−streptomycin at 37 °C in a 5% CO2 humidified air environment. MDA-MB-231 were maintained in Dulbecco’s modified eagle medium (DMEM) at 37 °C in a 5% CO2 humidified air environment. The medium was supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v) penicillin−streptomycin. Hyperaggressive ovarian cancer cells ID8-B7 were maintained in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated FBS, 2 mM Lglutamine, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, and 1× 100 U penicillin−streptomycin. In Vitro DOX Internalization Studies. Confluent cells were removed using 0.05% (w/v) trypsin−EDTA and seeded into a glass bottom disposable culture dish at a density of 2.5 × 105 cells in 2 mL of complete medium. The cells were incubated overnight at 37 °C in a 5% CO2 humidified air environment. Following overnight incubation, Hoechst 34580 dye for live-cell imaging was added to cells to enable visualization of cell nuclei. Live cells were imaged at 37 °C in a 5% CO2 humidified air environment. 405 and 488 nm solidstate diode lasers were used to excite the Hoechst 34580 and DOX, and corresponding emission from each was collected (405 nm, intensity = 10%, 415−475 nm emission monitored; 488 nm, intensity = 10%, 540−620 nm emission monitored). After the first image was collected, DOX or copolymer−DOX corresponding to a concentration of 1.5 μM DOX was added to the culture dish without disrupting the z-plane of the cells. A confocal image was acquired every 20 min for a total of 24 h, and the sample was monitored every 4−6 h to ensure that the cells had not drifted from the imaging plane. Cytotoxicity Studies. Confluent cells were removed using 0.05% (w/v) trypsin−EDTA and were seeded (2 × 103 cells in 100 μL complete medium) in a sterile, tissue culture treated, 96-well, clear bottom plate and incubated at 37 °C in a 5% CO2 humidified air environment overnight. Following overnight incubation, cells were washed 2 times with PBS and incubated for 24 h with free DOX at concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 μM, DOX−copolymer at concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 25 μM, and a copolymer control corresponding to the highest concentration of copolymer used in 25 μM DOX−copolymer (n = 6). After 24 h, cells were washed twice with PBS to remove free drug, the medium was replaced with fresh medium, and cells were returned to the

recrystallized from hexanes prior to use. All other chemicals were used as received. Sephadex G-25 M with 0.15% Kathon CG filtration columns were a generous gift from the Case Western Reserve University Department of Dermatology. Instrumentation. 1H NMR and 19F NMR spectra were taken on a 600 MHz Varian Inova NMR spectrometer with residual solvent peaks used as a reference for all NMR spectra. ESI-MS was acquired using a Thermo LCQ DECA. UV−vis spectra were acquired on a PerkinElmer Lambda 800 UV−vis. Gel permeation chromatography (GPC) was performed using a Tosoh EcoSEC-HCL-83200-GPC using THF as the elution solvent. Dynamic light scattering (DLS) was conducted on a Wyatt DynaPro NanoStar. The MTT assay was completed using a Biotek Synergy HT microplate reader. Confocal microscopy was performed on a Leica TCS SPE confocal microscope using a 63× oil immersion objective. Synthesis of Hydrazone−DOX−Alkyne. Doxorubicin hydrochloride (0.013 g, 0.02 mmol) was dissolved in 5 mL of dry MeOH in an oven-dried round-bottom flask equipped with a stir bar. To the reaction vessel, was added pent-4-yne hydrazine (0.004 g, 0.04 mmol, see Supporting Information for synthesis) followed by 10 μL of trifluoroacetic acid. The reaction proceeded at room temperature for 18 h under a nitrogen atmosphere. After 18 h, the excess solvent was removed in vacuo, yielding a deep red oil. The oil was dissolved in approximately 5 mL of methanol, washed three times with 5 mL of hexanes, and dried again, yielding a deep red oily solid (0.012 g, 79%). ESI-MS in methanol: calculated for DOX− alkyne, C32H35N3O11 [M + H] 638.2 m/z, found 638.8 m/z; DOX−HCl−alkyne, C32H36ClN3O11 [M + K] 712.2 m/z, found 712.1 m/z. Analysis of the 1H NMR spectrum revealed formation of hydrazone−DOX−alkyne (Figure S2). Synthesis of DOX−Copolymer. Copolymer 2 (0.013g, 2.3 μmol) was synthesized as previously described46 and dissolved in 1 mL of a 1:1 methanol:water solution in a vial equipped with a stir bar. The hydrazone−DOX−alkyne (0.003 g, 4.7 μmol) was dissolved in 1 mL of dry methanol and added to the reaction mixture. The following aqueous solutions were subsequently added to the reaction mixture: a premixed solution of CuSO4 (10 μL, 10 mM) and THPTA (5 μL, 50 mM) and then a freshly made solution of sodium ascorbate (20 μL, 100 mM). The reaction proceeded for 30 min at room temperature; after 30 min a fresh solution of sodium ascorbate was added to the reaction mixture, and the reaction proceeded for another 30 min. The reaction mixture was dried in vacuo, and the resulting product was reconstituted in 1 mL of 1% methanol in water and purified via a gravity-fed size exclusion column (Pharmacid) packed with Sephadex G-25 M with 0.15% Kathon CG in distilled water using a 1% methanol solution. The pure material was dried in vacuo (0.015 g, 79% recovery). Analysis of the 1H NMR spectrum revealed resonance peaks corresponding to polymer and DOX (Figure S3). Determination of DOX Conjugation Yield. The concentration of DOX covalently attached to the copolymer was determined using UV/visible spectroscopy and the Beer− Lambert law. A concentration curve with 6 concentrations of the DOX−copolymer conjugate dissolved in deionized water was generated. The absorbance at 481 nm was monitored as it corresponds to the λmax of DOX with a reported molar absorptivity coefficient (ε = 11,400 M−1 cm −1).48 Using these values, it was determined that an average of 0.65 mol of DOX was present per mol of conjugate. C

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Figure 1. Characterization of 19F-DOX 3. (A, B) Gel permeation chromatography (GPC) trace monitoring the refractive index (RI) (solid black line) and the UV absorbance at 252 nm (dashed red line) of (A) copolymer 2 and (B) DOX−copolymer. (C, D) Dynamic light scattering (DLS) of (C) copolymer 2 dissolved in ultrapure water and (D) 19F-DOX 3 dissolved in ultrapure water.

incubator for 24 h. Cell viability was analyzed using an MTT assay. Wells were aspirated and MTT solution was added (100 μL/well, 85:15 (v/v) complete DMEM:MTT solution, 5 mg/ mL in PBS), and incubation was continued until purple formazan crystals appeared, approximately 4 h. Medium was aspirated and DMSO was added (100 μL/well) to dissolve formazan crystals. The plate was placed on an orbital shaker for 30 min in the dark at 50 rpm. The absorbance of the dissolved formazan was analyzed at 620 nm using a Biotech Synergy HT microplate reader. Cell viability was determined by normalizing treated cells as a percentage relative to the untreated cells. Values are presented as averages ± standard deviation, n = 6.

The DOX−alkyne was conjugated to the azide-terminated random copolymer using the ligand-accelerated CuAAC reaction, employing a catalyst system of THPTA, sodium ascorbate, and copper sulfate.50 This catalyst system has previously been used to accelerate the reaction by lowering activation energy and, for this copolymer system, has enabled the attachment of various cargoes to the copolymer.46,47 The click reaction was carried out in a mixed solvent system of equal parts methanol and water to allow full dissolution of the DOX− alkyne. The crude reaction mixture was purified with a gravityfed size exclusion column to yield the pure product. Analysis by gel permeation chromatography (GPC) coupled with a UV detector determined that no unconjugated DOX was present in the sample (Figure 1B). Additionally, an increase in the molecular weight was observed with the covalent attachment of the DOX−alkyne (Mn of 2 ∼ 10.5 kDa, Mn of 3 ∼ 11.1 kDa) consistent with the covalent attachment of a single DOX molecule. The dispersity of the DOX−copolymer increased slightly (Đ of 2 = 1.1, Đ of 3 = 1.3), likely a result of incomplete conjugation yield. Following GPC, nuclear magnetic resonance (NMR) spectroscopy confirmed proton resonance peaks characteristic of DOX, δ 7.7, 7.4 ppm, while the same proton resonances from the copolymer components were observed, δ 4.3, 4.0, 3.6 ppm (Figure S3). DOX concentration and effective yield were determined by UV/vis absorbance at the λmax (481 nm) as compared to the NMR-derived molecular weight of the copolymer and indicated that an average of 0.65 mol of DOX was attached per DOX conjugate (Figure S4). Highly fluorinated molecules are known to self-assemble into micelles, and in our past work these polymers formed micelles of the appropriate size to harness the enhanced permeability and retention (EPR) effect. The size of the conjugate was determined using dynamic light scattering (DLS) to ensure that DOX conjugation had minimal impact on the polymer’s ability to self-assemble into micelles. The conjugate was dissolved directly into ultrapure water and vortexed for approximately 30

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RESULTS AND DISCUSSION Conjugate Synthesis and Characterization. In our synthetic scheme, we sought to employ a functional group that was known to be pH-responsive for cancer drug delivery while also using a high yielding reaction to install DOX onto the polymer backbone. Since the ligand-accelerated coppercatalyzed azide−alkyne cycloaddition (CuAAc) reaction is of high fidelity and often high yielding at low concentrations, we chose to utilize this reaction to couple these high-value materials. Therefore, we synthesized a hydrazone linked DOX appended with an alkyne handle.27 Hydrazone-linked DOX− alkyne 1 was formed through three synthetic steps (Scheme S1). Briefly, tert-butyl carbazate was coupled to 4-pentynoic acid through a DCC mediated reaction to form a tertbutyloxycarbonyl (Boc) protected hydrazine alkyne. The Boc deprotection was achieved using a solution of 4 N HCl in dioxanes to form the hydrazine−alkyne. The hydrazine−alkyne reacted with the carbonyl on doxorubicin hydrochloride via an acid catalyzed condensation reaction to afford the DOX− hydrazone−alkyne (1), a result confirmed by ESI-MS and 1H NMR (Figure S3). This hydrazone linkage is pH-sensitive as it cleaves in mildly acidic conditions.49 D

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efficacy of anticancer drugs against ovarian cancer and can develop drug resistance with continued exposure to chemotherapeutics like cisplatin. ID8 cells were chosen because they mimic aggressive metastatic disease and in the future represent an excellent in vivo model. Lastly, MDA-MB-231 cells were chosen as a model for triple negative breast cancer, as these cells are a well-studied model that is DOX responsive. These in vitro cell models have been widely used to probe drug delivery efficacy51 and have been utilized in well established in vivo models.52 Internalization into the cell is necessary to facilitate the pHtriggered release of DOX from the carrier via the pH of the lysosomal compartment and/or endosomes. Confocal microscopy was used to probe the time dependent uptake of the drug into the cancer cell lines. Live cell imaging was carried out over a 24 h period. Cells were stained with Hoechst dye to visualize live cell nuclei and monitored over time after the addition of DOX (5.0 μM). DOX was visualized by the molecule’s own intrinsic fluorescence. In general, the fluorescent signal from free-DOX and 19F-DOX appears inside of the cells 2 h after the drug has been added to the cell solution, with increasing DOX signal intensity observed over time indicating increased drug accumulation. Copolymer−DOX shows a punctate fluorescence distribution within all cell lines, likely indicative of endosomal internalization, with a small amount of drug visible in the nucleus. In the A2780 cell line, internalization of DOX begins around 2 h (Figure 3B), and after 12 h (Figure 3C) the fluorescent

s to facilitate dissolution and promote micelle assembly. The hydrodynamic diameter of the conjugate was determined to be 250 nm ±14 nm from concentrations ranging from 1.5 mM (10 mg mL−1) to 3.0 μM (0.02 mg mL−1) (Figures 1D, S5). 19FDOX has a hydrodynamic diameter that is approximately the same size as copolymer 2 (260 nm ±16 nm, Figure 1C). The size of nanoparticles is critically important for delivery to solid tumors by passive tumor targeting, and these results verify that DOX conjugation has minimal impact on hydrodynamic volume. Furthermore, once 19F-DOX is diluted it still maintains its ability to form micelles at concentrations as low as 0.02 mg mL−1, which may be important for dilution effects in future in vivo experiments. The release profile of DOX from the copolymer prodrug 3 was probed by separating free drug from the conjugate by dialysis and monitoring the absorbance of released drug from the carrier. The copolymer was dissolved and dialyzed against PBS at pH 7.4 or sodium acetate buffer at pH 5.0 for 48 h with released aliquots collected periodically. These conditions were chosen to mimic the pH of lysosomal and tumor microenvironments compared to the physiological pH of blood. Cumulative percent release of DOX from the copolymer in acidic medium was more rapid with more drug released after the 48 h time period (21 ± 1.4%) than in neutral pH (7.4 ± 3.6%) (Figure 2). In addition, within the first 4 h of incubation

Figure 2. Release of DOX from 19 F-DOX. (A) Schematic representation of pH triggered release. (B) Percent cumulative release of DOX from the copolymer carrier over time from sodium acetate buffer pH 5.0 (red circles) and PBS pH 7.4 (blue circles).

in the acidic environment, burst release was greater (8.3 ± 2.2%) than in neutral pH (2.3 ± 1.0%). These results indicate pH sensitivity of the prodrug consistent with the acid-labile hydrazone linkage.49 Of note, cumulative release is likely vastly underestimated since DOX can be readily incorporated into the polymer micelles noncovalently even upon hydrolysis of the hydrazone bond. These results do however further demonstrate that the 19F-DOX could serve to selectively deliver DOX to acidic microenvironments while reducing premature drug release during circulation. In Vitro Cell Studies. The therapeutic potential of DOX− copolymer was evaluated using three ovarian cancer cell lines and one triple negative breast cancer model. These tumor types are clinically treated with DOX and have no validated molecular signatures, hence we felt that efficacy with an untargeted particle would be of utmost value in delivering DOX. In vitro experiments with human ovarian endometroid adenocarcinoma (A2780), human ovarian adenocarcinoma (OVCAR3), murine hyper aggressive metastatic (ID8) ovarian disease, and triple negative breast cancer (MDA-MB-231) were carried out to evaluate internalization and toxicity of 19F-DOX. A2780 and OVCAR3 are human cell lines commonly used to evaluate the

Figure 3. Representative confocal images from time lapse of DOX (A−D) and 19F-DOX (E−H) uptake in A2780 cells. (A−D) DOX uptake at t = 0 (A), 2 h (B), 12 h (C), and 24 h (D). (E−H) 19F-DOX uptake at t = 0 (E), 2 h (B), 12 h (G), and 24 h (H). Scale bar is 50 μm.

signal from DOX is seen dispersed throughout the cell with no observable increase at the 24 h time point (Figure 3D). Internalization of the 19F-DOX into A2780 follows a similar trend as the DOX, with fluorescence signal from DOX visible after 2 h (Figure 3F) with a slight increase in the fluorescent signal at 12 h (Figure 3G), and after 24 h of incubation with 19 F-DOX there appears to be fluorescent signal dispersed throughout the cells (Figure 3H). These results indicate that both DOX and 19F-DOX can internalize into A2780 cells, however it is clear that the image intensity of DOX is lower once attached to the polymer. The results from the internalization into the OVCAR3 cell line are similar to the A2780 internalization. For cells incubated with DOX, the fluorescent signal appears 2 h (Figure 4B) after E

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DOX, after 2 h the fluorescence signal is seen across the nucleus (Figure 6B), and after 12 h the intensity of the DOX

Figure 4. Representative confocal images from time lapse of DOX (A−D) and 19F-DOX (E−H) uptake in OVCAR3 cells. (A−D) DOX uptake at t = 0 (A), 2 h (B), 12 h (C), and 24 h (D). (E−H) 19F-DOX uptake at t = 0 (E), 2 h (B), 12 h (G), and 24 h (H). Scale bar is 50 μm.

Figure 6. Representative confocal images from time lapse of DOX (A−D) and 19F-DOX (E−H) uptake in MDA-MB-231 cells. (A−D) DOX uptake at t = 0 (A), 2 h (B), 12 h (C), and 24 h (D). (E−H) 19 F-DOX uptake at t = 0 (E), 2 h (B), 12 h (G), and 24 h (H). Scale bar is 50 μm.

the addition of drug with a steep increase in fluorescent intensity, indicating more drug uptake after 12 h (Figure 4C), and after 24 h fluorescence from DOX is extremely intense (Figure 4D). The internalization of 19F-DOX into cells is visible after 2 h as punctate, localized fluorescence (Figure 4F); after 12 h, the signal increases and is localized near the nucleus (Figure 4G); and after 24 h of incubation with DOX− copolymer some cell nuclei appear to shrink and the fluorescent signal appears equivalent to that at the 12 h time point. For the ID8 cells incubated with DOX and 19F-DOX, the fluorescent signal from DOX is less intense than in the other ovarian cancer cell lines (Figure 5). For cells incubated with the

signal has further increased, indicating more drug uptake (Figure 6C), while at 24 h the intensity of the fluorescence signal has not increased (Figure 6D). In cells incubated with 19 F-DOX, the fluorescence signal appears after 2 h localized outside of the nucleus visualized as punctate fluorescent signals (Figure 6F). After 12 h, the 19F-DOX-signal intensity increases with more signals appearing around the nucleus and in the nucleus (Figure 6G) indicating increased drug uptake. After 24 h, the fluorescence signal appears to increase compared to the image taken after 12 h (Figure 6H), indicating increase uptake of 19F-DOX. To evaluate cancer-killing efficacy, a dose−response curve was generated for cell lines using an MTT assay (Figure 7). Both DOX and 19F-DOX were incubated with cancer cell lines at eight concentrations of DOX and evaluated using an MTT assay to compare the cell viability of treated cells to a nontreated control. The IC50 results from the MTT assays are summarized Table 1. A control using the highest concentration of copolymer in the 19F-DOX did not induce cell killing in any of the cell lines, indicating that cell death was caused by DOX (Table S2). In A2780 cell line, the free DOX exhibits a greatly enhanced IC50 of 0.05 μM when compared with the 19F-DOX, which demonstrates an IC50 of 1.6 μM. Likewise for OVCAR3, an IC50 value for DOX of 0.4 μM was observed while the copolymer has a value of 4.4 μM to achieve the same cell killing. This difference in efficacy between free DOX and 19FDOX is not unexpected, as these trends have been previously observed between free drug and carrier in synthetic nanoparticles and viral carriers.43,53,54 The difference in efficacy is likely correlated to internalization efficiency. DOX is taken up more rapidly by cells via passive diffusion across the cell membrane, while internalization of DOX−copolymer likely requires endocytosis or macropinocytosis due to the size of the particle.55 In addition, it is possible that uptake via these mechanisms may not fully release DOX until a late endosome or endolysosome stage is reached and a concomitant very low pH is achieved, thus limiting the delivery of the drug to the nuclei, which is essential in order for DOX to act on topoisomerase I and II.

Figure 5. Representative confocal images from time lapse of DOX (A−D) and 19F-DOX (E−H) uptake in ID8-B7 cells. (A−D) DOX uptake at t = 0 (A), 2 h (B), 12 h (C), and 24 h (D). (E−H) 19F-DOX uptake at t = 0 (E), 2 h (B), 12 h (G), and 24 h (H). Scale bar is 50 μm.

sample of DOX and 19F-DOX, the DOX signal is more localized within and around the cell nucleus after 12 h (Figures 5C and 5G). At 24 h, fluorescence signal from DOX is visible throughout the nucleus of cells for both samples, as evidenced by merged images exhibiting a purple color in the nucleus. From the images there is no apparent difference in uptake between the two samples, which is also reflected in the efficacy data. Drug internalization in the breast cancer cell line, MDA-MB231, shows trends similar to the A2780 and OVCAR3. After 2 h, fluorescence signal from DOX emerges in cells incubated with both DOX and 19F-DOX. In cells incubated with free F

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Figure 7. Cytotoxicity of DOX and 19F-DOX. IC50 curves generated from MTT assays for ovarian cell lines, (A) A2780, (B) OVCAR3, and (C) ID8, and breast cancer cell line, (D) MDA-MB-231, following 24 h incubation with either DOX or 19F-DOX.

and aqueous conditions with tolerance to various functional groups.

Table 1. IC50 Values for DOX and DOX−Copolymer for Various Cancer Cell Lines cell line

DOX IC50 [μM]

A2780 OVCAR3 ID8-B7 MDA-MB-231

0.05 0.4 0.55 1.8

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19

F-DOX IC50 [μM]

CONCLUSIONS The chemotherapeutic DOX was decorated with an alkyne via a pH-sensitive hydrazone linkage and covalently attached to the azide terminus of a low molecular weight fluorous copolymer carrier using the azide−alkyne cycloaddition. The copolymer− DOX assembles into micelles with a hydrodynamic diameter of approximately 250 nm in water with DOX release demonstrated in acidic aqueous media. In vitro results comparing the free drug to the 19F-DOX using two human derived ovarian cancer cell lines, A2780 and OVCAR3, one murine model, ID8, and a triple negative breast cancer model show that 19F-DOX is readily internalized and maintains efficacy. These results are highly cell-line dependent and can be readily correlated to internalization efficiency and localization of DOX within the cell. These results indicate that a fluorous carrier can effectively be decorated with an alkyne−hydrazone−DOX using the CuAAc reaction creating stable micelles capable of efficacy across a wide panel of difficult to treat in vitro cancer models.

1.6 4.4 0.35 1.2

Treatment of murine derived ovarian cancer ID8 cells with DOX and 19F-DOX shows IC50 values that are approximately equivalent with 19F-DOX showing a slightly lower IC50 concentration than DOX (0.35 μM versus 0.55 μM). This result correlates well to the confocal micrograph (Figure 5) indicating that DOX and 19F-DOX are internalized into the nucleus of these cells after 24 h incubation. In the breast cancer cell line, MDA-MB-231, IC50 values following treatment with DOX and 19F-DOX are similar to 19F-DOX having a slightly lower IC50 value than free DOX (1.2 μM versus 1.8 μM). In all cases, both DOX and the 19F-DOX effectively killed cancer cells, with the DOX−copolymer demonstrating a similar inhibitory concentration as other nanoparticle−DOX carriers.30,56 In the case of MDA-MB 231 cells, internalization efficiency between DOX and 19F-DOX did not strictly correlate with efficacy, and further studies will investigate the cellular mechanism behind this phenomenon. The design of this system is intended to deliver the drug selectively within the tumor microenvironment or within the lysosome or endosome to mitigate toxicity. The copolymer micelle has previously demonstrated exceptional uptake into solid tumors via passive tumor targeting and rapid clearance in vivo further perpetuating the expected therapeutic viability for drug delivery in cancer treatment. The azide end unit of the copolymer provides a functional handle to attach alkyne-functionalized cargo, like a pH-sensitive DOX, using the CuAAC reaction. The click reaction is an invaluable tool in any synthetic chemistry toolbox as it is broad in scope, high yielding, rapid, and occurs in mild

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b01046. DOX-alkyne synthesis and characterization, DLS, and cytotoxicity results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jonathan K. Pokorski: 0000-0001-5869-6942 G

DOI: 10.1021/acs.molpharmaceut.7b01046 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NIH R21 HL121130 to J.K.P. We thank Dr. Yinghua Chen and the Protein Expression Purification Crystallization and Molecular Biophysics Core in the Department of Physiology and Biophysics (Case Western Reserve University) for assistance with DLS measurements.

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DOI: 10.1021/acs.molpharmaceut.7b01046 Mol. Pharmaceutics XXXX, XXX, XXX−XXX