Using Peptide Aptamer Targeted Polymers as a Model Nanomedicine

Sep 7, 2017 - (15, 16) In addition to therapeutic benefits, its unique stability and intrinsic fluorescence properties make DOX an ideal model drug fo...
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Using Peptide Aptamer Targeted Polymers as a Model Nanomedicine for Investigating Drug Distribution in Cancer Nanotheranostics Yongmei Zhao, Zachary H. Houston, Joshua D. Simpson, Liyu Chen, Nicholas L. Fletcher, Adrian V. Fuchs, Idriss Blakey, and Kristofer J. Thurecht* Centre for Advanced Imaging, Australian Institute for Bioengineering and Nanotechnology, and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of Queensland, Brisbane, 4072, Australia S Supporting Information *

ABSTRACT: Theranostics is a strategy that combines multiple functions such as targeting, stimulus-responsive drug release, and diagnostic imaging into a single platform, often with the aim of developing personalized medicine.1,2 Based on this concept, several well-established hyperbranched polymeric theranostic nanoparticles were synthesized and characterized as model nanomedicines to investigate how their properties affect the distribution of loaded drugs at both the cell and whole animal levels. An 8-mer peptide aptamer was covalently bound to the periphery of the nanoparticles to achieve both targeting and potential chemosensitization functionality against heat shock protein 70 (Hsp70). Doxorubicin was also bound to the polymeric carrier as a model chemotherapeutic drug through a degradable hydrazone bond, enabling pH-controlled release under the mildly acid conditions that are found in the intracellular compartments of tumor cells. In order to track the nanoparticles, cyanine-5 (Cy5) was incorporated into the polymer as an optical imaging agent. In vitro cellular uptake was assessed for the hyperbranched polymer containing both doxorubicin (DOX) and Hsp70 targeted peptide aptamer in live MDA-MB-468 cells, and was found to be greater than that of either the untargeted, DOX-loaded polymer or polymer alone due to the specific affinity of the peptide aptamer for the breast cancer cells. This was also validated in vivo with the targeted polymers showing much higher accumulation within the tumor 48 h postinjection than the untargeted analogue. More detailed assessment of the nanomedicine distribution was achieved by directly following the polymeric carrier and the doxorubicin at both the in vitro cellular level via compartmental analysis of confocal images of live cells and in whole tumors ex vivo using confocal imaging to visualize the distribution of the drug in tumor tissue as a function of distance from blood vessels. Our results indicate that this polymeric carrier shows promise as a cancer theranostic, demonstrating active targeting to tumor cells with the capability for simultaneous drug release. KEYWORDS: theranostic nanoparticles, doxorubicin, Hsp70 targeted aptamer, cyanine-5, simultaneous drug release, drug delivery

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INTRODUCTION Multifunctional theranostic nanoparticles (NPs),3,4 especially polymeric NPs, have been widely investigated due to their excellent biocompatibility, biodegradability, and diverse functionality.5−7 However, many challenges still remain before mainstream utilization of nanoparticles is possible. For example, liposomes can become destabilized during vesicle fusion,8 while macromolecular species such as dendrimers usually require multistep synthetic and purification steps, which significantly increases the time to production and decreases economic viability.9 Thus, there is a significant need to develop a new platform to overcome these limitations and provide a low cost, highly stable, and functionalizable drug delivery system. Hyperbranched polymers have a highly branched architecture with the potential for expressing multiple functionalities, which enables the combination of both diagnostic and therapeutic agents within the same molecule.10 Moreover, hyperbranched © XXXX American Chemical Society

polymers are very simple to synthesize via a one-pot RAFT polymerization. We have previously shown that hyperbranched polymers composed of a poly(ethylene oxide) backbone exhibit excellent biocompatibility and long circulation in vivo.11,12 Thus, hyperbranched polymers provide an ideal theranostic platform to develop next generation drug delivery systems. Doxorubicin (DOX) is a widely used clinical chemotherapeutic drug owing to its mechanism of action through intercalation with DNA and inhibition of macromolecular biosynthesis;13,14 however, its toxicity is not limited toward cancer, and it exhibits significant side effects, the most common of which is cardiotoxicity.15,16 In addition to therapeutic Received: July 2, 2017 Revised: August 21, 2017 Accepted: August 24, 2017

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DOI: 10.1021/acs.molpharmaceut.7b00560 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics benefits, its unique stability and intrinsic fluorescence properties make DOX an ideal model drug for both in vitro and in vivo diagnostic studies. Thus, there are many examples in the literature describing the use of DOX as the therapeutic of choice in nanomedicine.17−19 Among them, pH-sensitive polymeric nanoparticles have shown high potency in the delivery of DOX,17 because they rely on well-established pH gradients in solid tumors (pH 6.5−7.2)20,21 and in the endosomal/lysosomal compartments of tumor cells (pH 4.0− 6.5).22 In order to take advantage of these intratumoral pH gradients, it is necessary to deliver higher concentrations of the drug to the tumor site and then monitor the drug release and cellular distribution following internalization into cells of interest. Heat-shock protein 70 (Hsp70) is a protein that plays an important role in normal cellular machinery by assisting in protein folding, while protecting cells from stressful variations in the microenvironment such as a lack of nutrients or oxygen.23−25 It has also been found to be overexpressed in many types of tumors such as breast,26 colon,27 and malignant melanoma.28 These findings have led to numerous studies to develop Hsp70 as a biomarker or a putative target for cancer therapy.29,30 Among these studies, Rérole and his group investigated a small peptide aptamer which had high affinity for Hsp70.31 Based on this work, Coles et al. developed a targeted nanomedicine using this 8-mer peptide aptamer as a targeting moiety and showed that it had greater tumor specificity than a similar polymer targeted to the folate receptor using optical imaging.12 An important consideration is that this peptide aptamer does not induce cytotoxicity on its own but rather has shown strong chemosensitization when used in parallel with general chemotherapeutic drugs such as cisplatin or etoposide.31 This work uses Hsp70 as both a targeting and potential chemosensitizing agent for breast cancer therapy using the established chemotherapeutic, DOX. DOX was bound to the polymeric carrier utilizing a degradable hydrazone bond, enabling pH-controlled release under mildly acidic conditions that are present in endosomes/lysosomes of tumor cells. A fluorescent dye (Cy5) was bound to the hyperbranched polymer in order to track the biodistribution and intracellular distribution of both the DOX and the nanocarrier separately in both in vitro and in vivo analysis. Thus, here we report on the cellular delivery of a theranostic based on a hyperbranched polymer scaffold.

use. Ultrapure water (18.2 mΩ/cm) was obtained from an Elga ultrapure water system. All products that related to cell biology including cell culture media Dulbecco’s modified Eagle medium (DEME), RPMI1640, fetal bovine serum, (FBS), penicillin−streptomycin antibiotic solution, trypsin, trypan blue solution, and phosphate saline buffer (PBS) were purchased from Sigma, USA. Cell lines MDA-MB-231 and MDA-MB-468 were both incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. Characterization. 1H NMR spectra were acquired on either a Bruker Avance 400 or 500 MHz spectrometer. A gel permeation chromatography multiangle laser light scattering (GPC-MALLS) system consisting of a 1515 isocratic pump (Waters), Styragel HT 6E and Styragel HT 3 columns (Waters), 2414 differential refractive index detector (Waters), and a Dawn Heleos laser light scattering detector (Wyatt) with THF as an eluent at a flow rate of 1 mL/min was used to measure the molecular weight and dispersity of the polymers. Dynamic light scatting (DLS) was used to measure the hydrodynamic diameter using a Malvern Zetasizer. Synthesis of N-(tert-Butoxycarbonyl)-N′-(6methacrylamidohexanoyl)hydrazine (TBMC) Monomer. To a solution of tert-butyl carbazate (2.5 g 0.018 mol) and pyridine (6 mL) in 10 mL of DCM was added methacrylic anhydride (4.4 g 0.028 mol) dropwise over 30 min at 0 °C. After 10 min, the mixture became homogeneous, and stirring was continued for 16 h at room temperature. The reaction mixture was then washed with saturated sodium bicarbonate, again with saturated sodium chloride, dried over magnesium sulfate, and concentrated to give the crude product. The mixture was then purified by silica gel flash chromatography using a 10% methanol in chloroform eluent to give the pure product as a white solid, 2.27 g (60%). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.49 (s, 9H, CH3), 2.00−2.02 (m, 3H, CH3), 5.45−5.47 (m, 1H, CHH), 5.78 (s, 1H, CHH), 6.60 (s, 1H, NHNH), 7.60 (s, 1H, NHNH). HRESI(+)MS m/z: 223.1065 [M + Na]+ (calcd for C9H16N2O3, 223.1053). HRESI(+)MS m/z: 423.2230 [2M + Na]+. Synthesis of RAFT Agent 4-Cyano(dodecylsulfanylthiocarbonyl)sulfanyl Pentanoic Propyne (Alk-RAFT). A 100 mL, 3-neck round bottomed flask fitted with a reflux condenser, solids addition port, thermowell, and stirrer bar was charged with bis(dodecylsulfanylthiocarbonyl) disulfide (1.7 g, 1.51 mmol) and 40 mL of ethyl acetate. The resulting solution was heated to gentle reflux and reacted with 4,4′-azobis(4-cyanopentanoic acid) (1.4 g, 5.14 mmol) over 3 h. The reaction mixture was heated for an additional 19 h. Ethyl acetate was removed under reduced pressure, and the product was allowed to crystallize from heptane. The solid was filtered, washed with water, and dried to provide 4-cyano(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid as a yellow solid (2.2 g, yield 91%). To a solution of 4-cyano(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid (1 g, 2.34 mmol), DMAP (28.6 mg, 0.23 mmol), and propargyl alcohol (262 mg, 4.68 mmol) in DCM was added EDC·HCl (897 mg, 4.68 mmol) in 10 mL of DCM dropwise at 0 °C. After 10 min the mixture became homogeneous and was stirred at room temperature for 12 h. Then the reaction mixture was filtered and the organic layer washed with saturated sodium chloride, dried over anhydrous magnesium sulfate, and concentrated. The crude product was then purified by silica gel flash chromatography using a 10% ethyl acetate in hexane solvent system to give the pure product

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EXPERIMENTAL SECTION Materials. Doxorubicin hydrochloride, trifluoroacetic acid (TFA), dicyclohexylcarbodiimide (DCC), methacryloyl chloride, tert-butyl carbazate, 4-(dimethylamino)pyridine (DMAP), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC·HCl), and 4,4′-azobis(4-cyanopentanoic acid) were bought from Sigma-Aldrich and used directly without any purification. Cyanine-5 amine was purchased from Lumiprobe. Azobis(isobutyronitrile) (AIBN, Sigma-Aldrich) was recrystallized twice from methanol before use. Solvents including nhexane, ethyl acetate, dichloromethane (DCM), dimethylformamide (DMF), diethyl ether, pyridine, tetrahydrofuran, acetonitrile, and methanol were used dry where applicable and of reagent grade quality. Poly(ethylene glycol methacrylate) (PEGMA, MW = 475g·mol−1) and ethylene glycol dimethacrylate (EGDMA) were purified to remove radical inhibitors by passing through a basic alumina column before B

DOI: 10.1021/acs.molpharmaceut.7b00560 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics as a yellow liquid (0.9 g 82%) (see Figure S1 for the 1H NMR and 13C NMR). ESI-MS (positive mode, methanol): calcd [M]+ 441.18 g/mol; measured [M + Na]+ 464.32. Synthesis of Cyanine-5 Methacrylamide (Cy5-MA). Cy5-MA monomer was incorporated into HBP-1 during polymerization as a fluorescent imaging agent to track the polymer in vitro and in vivo. Detailed synthesis of the monomer is described in previous work.32 Synthesis of Poly(PEGMA-co-TBMC-co-EDGMA-coCy5MA) (HBP-1). PEGMA (avg Mn 475, 500 mg, 1.05 mmol), TBMC (53 mg, 2.63 × 10−1 mmol), Cy5-MA (3 mg, 0.0046 mmol), EGDMA (13 mg, 6.5 × 10−2 mmol), AIBN (2.8 mg, 1.71 × 10−2 mmol), Alk-RAFT (29 mg, 6.5× 10−2 mmol), and 900 μL of dry tetrahydrofuran (THF) were placed in a 10 mL Schlenk flask equipped with a magnetic stirrer bar, sealed with a silicone septum and reinforced with a cable tie, and the reaction mixture was bubbled with nitrogen for 10 min. The Schlenk flask was placed in an oil bath and stirred at 75 °C for 24 h. The reaction mixture was then precipitated from n-hexane three times, purified via size-exclusion chromatography (SEC), with a 20% EtOH/H2O eluent, and lyophilized to give the pure polymer as a blue oil (95% conversion, 543 mg). Diagnostic peaks from 1H NMR (500 MHz, CDCl3): 1.35 ppm (s, tBu), 3.99 ppm (s, COOCH2 PEGMA), 4.58 ppm (CH2CHCH RAFT). GPC-MALLS: 34000 g/mol; Đ = 1.67. 13.0 TBMC monomer units per polymer. Deprotection of tert-Butyloxycarbonyl Group (HBP-2). Deprotection of the tert-butyloxycarbonyl group was achieved by addition of 20% TFA (400 μL) in DCM (1.6 mL) to HBP1, and the reaction mixture was stirred under reflux for 6 h. The reaction mixture was then dissolved in a minimal amount of THF and precipitated from n-hexane three times. The crude oil was then further purified by SEC. After purification, the polymer was lyophilized for 24 h to give the pure product as a blue solid in quantitative yield. Diagnostic peaks from 1H NMR (500 MHz, CDCl3): 4.01 ppm (s, COOCH2 PEGMA), 4.62 ppm (CH2CHCH RAFT). The amount of hydrazide groups remaining after deprotection was determined by the TNBSA assay (2,4,6trinitrobenzenesulfonic acid), which is a colorimetric assay for determining amine concentration in solution. This assay suggested that there were 10.4 amines per HBP. This suggests that minimal degradation of the methacrylamide was observed during removal of the t-butyl groups through deprotection. Attachment of Aptamer Targeting Moiety (HBP-3). HBP-2 (25 mg, 2.1 × 10−5 mol end groups) was dissolved in 50% DMSO/H2O (1.5 mL). Copper sulfate (1 mg, 6 × 10−6 mol), ascorbic acid (2.5 mg, 1.2 × 10−5), tris[(1-benzyl-1H1,2,3-triazol-4-yl)methyl]amine, and the aptamer (N-terminal (5-azidopentanoic acid)-SPWPRPTY) (2.8 mg, 2.2 × 10−5 mol) were added, and the solution was degassed with nitrogen and stirred for 12 h at 60 °C. The reaction mixture was then directly purified by size-exclusion chromatography with 20:80 ethanol/H2O eluent and dried by lyophilization overnight to give final product. Diagnostic peaks from 1H NMR (500 MHz, CDCl3): 4.01 ppm (s, COOCH2 PEGMA), 5.11 ppm (CH2CHCH RAFT) (Figure S4). DOX Loading via Formation of Hydrazone Bond (HBP-4 and HBP-5). Either HBP-2 or HBP-3 (20 mg, 3.3 × 10−4 mmol) and DOX (2 mg, 3.6 × 10−3 mmol) were dissolved in 1 mL of anhydrous MeOH with a drop of glacial acetic acid. The reaction mixture was stirred in the absence of ambient light at reflux for 24 h. At the completion of the reaction, the excess

unreacted DOX was then removed by size-exclusion chromatography with MeOH as the eluent. After purification, the freeze-dried product was stored at −20 °C. The incorporation of doxorubicin was characterized by NMR, HPLC with a fluorescence detector (Figure S5), and UV−vis spectroscopy (Figure S7). None of the postmodification procedures following the synthesis of HBP-1 had a significant influence on the molecular weight or polydispersity of the hyperbranched polymer. Drug Release. The release of DOX from HBP-4 was investigated using reverse phase high performance liquid chromatography (HPLC) (column: CAPCELL PAKC18, type SG 120, 5 mm, size 4.6 mm × 250 mm with water−acetonitrile (0.1% TFA) gradient 20−100 vol % acetonitrile with flow rate of 1 mL/min) with fluorescent detection (excitation at 480 nm, and emission at 560 nm). First, the instrument was calibrated using a series of DOX concentrations in different sodium phosphate buffers (pH = 5 or 7.4). The amount of DOX released from the polymer conjugate was assessed after incubation in both pH = 5 and pH = 7.4 sodium phosphate aqueous solution at 37 °C via the reverse phase HPLC system at predetermined time intervals (Figure S6). In Vitro Cytotoxicity Analysis. The cytotoxicity of HBP-2, HBP-3, HBP-4, HBP-5, and free DOX against invasive human breast carcinoma cell lines MDA-MA-231 and MDA-MB-468 was investigated using the MTS assay (3-(4,5-dimethylthiazol2-yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium). First, cells with equal density (104 per well) were seeded in a 96-well plate and incubator for 24 h. Then, HBP-2, HBP-3, HBP-4, HBP-5, and free DOX were added at different concentrations in serum supplemented tissue culture medium after washing twice with PBS and were incubated for another 48 h. A second set of cells were kept without treatment to serve as a control group. After incubation, the culture medium was replaced with 100 μL of MTS solution (20 μL CellTiter 96 AQueous One Solution Reagent and 80 μL of tissue culture medium). Finally, the plates were incubated for one more hour before the absorbance was measured at 490 nm using a microplate reader. Using the cell viability of untreated cells (absorbance at 490 nm) as a benchmark, an IC50 was calculated by statistical software GraphPad Prism 6.0 using the relative fluorescence of the different treatment groups. Cellular Binding with Flow Cytometry. Flow cytometry was used to quantify cellular interaction following incubation of the cells with each treatment group. MDA-MB-468 cells were seeded in 6-well culture plates (2 × 105 cells/well). After preincubation for 24 h, HBP-2, HBP-3, HBP-4, HBP-5, free DOX, and a mixture of free aptamer (corresponding with the amount of the peptide attaching on the HBP-5) with HBP-3 were added at a concentration of 32 μg/mL. After 2 h, the cells were washed with PBS twice and lifted using Cellstripper (Media Tech, Inc.) and each well of cells was collected and labeled to analyze the cellular binding via FACSCalibur (fluorescence-activated cell sorting) flow cytometer (BD Bioscoience, USA). Each sample used 1 × 104 cells per experiment to analyze the fluorescence intensity via a fourdecade log scale fluorescence detector. To further investigate the aptamer targeting efficiency, a competitive binding assay was also undertaken, in which HBP at the same concentration as above was added to the cells after preincubating with 4.1 μg/ mL of free aptamer which was calculated according to the amount of the aptamer attaching to HBP-3 for 20 min. C

DOI: 10.1021/acs.molpharmaceut.7b00560 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Scheme 1. Synthetic Pathway for Development of the DOX-Loaded Theranostic Which Uses the Hyperbranched Polymer as a Scaffold

Confocal Laser Scanning Microscopy (CLSM) Study. MDA-MB-468 cells (1 × 105 cells per mL) were seeded onto Ibidi coverglass bottom μ-dishes (0.5 mL per dish) with 2 mL of RPMI-1640 medium supplemented with 10% FBS, which was added before the cells were returned to the incubator and left to reach confluence overnight. Cells were treated with 45 μg/mL of HBP-2, HBP-3, HBP-4, HBP-5, or free DOX (at a concentration of 2.7 μg/mL of DOX), and imaged using a Zeiss 710 laser scanning confocal microscope housed at the Australian Nanofabrication Facility Queensland Node. The Zeiss 710 was equipped with a 40× water immersion lens and argon and helium−neon lasers. Images were collected using a sequential scanning method, with Cy5 and DOX being collected in separate channels, excited with 633 and 488 nm lasers, respectively. Corresponding fluorescence data was collected between 643 and 700 nm and between 500 and 700 nm for each track. Images of cell populations were

collected at 15 min intervals with additional higher magnification images being retrieved between time points using the inbuilt digital zoom function. In order to examine the intracellular distribution of either the DOX or polymer variations, we utilized the region of interest (ROI) function inbuilt in Zen Lite 2012 (Blue Edition) software (Carl Zeiss Microscopy GmbH) and exported fluorescence intensity data to Microsoft Excel for analysis. Regions were selected via bright field images in order to minimize observer bias, and ROIs included the nucleus, nucleolus, vesicles, background, and cytoplasm, the latter being split into inner and peripheral cytosol components due to the presence of the endomembranous system being adjacent to the nucleus, this being an important area for endocytic processing and lysosomal interactions and also the location of important organelles.33 D

DOI: 10.1021/acs.molpharmaceut.7b00560 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics In Vivo Targeting Study. All studies were in accordance with guidelines of the Animal Ethics Committee of The University of Queensland, and the Australia Code for the Care and Use of Animals for Science Purposes. For all injections and imaging time points mice were anesthetized with 2% isoflurane in oxygen at a flow rate of 2 L min−1. Human breast cancer xenograft tumors were established in BALB/c nude mice (Australian Resources Centre, Australia) by injection of 4 × 106 MDA-MB-468 cells (50 μL of cold phosphate buffered saline, 27G needle) into the left inguinal mammary line, and tumor growth was monitored by caliper measurements twice per week. After 6 weeks of tumor growth, the mice were randomly assigned to the following 3 groups: saline (control, n = 1), HBP-4 (4.5 mg DOX kg−1, n = 6), and HBP-5 (4.5 mg DOX kg−1, n = 6). Live animal fluorescence imaging was performed at 24 and 48 h after injecting HBP-4 and HBP-5 into the tail vein of each group of mice. Ex vivo semiquantitative analysis, at 24 and 48 h, of the organs was also done to correlate with the in vivo data. In vivo and ex vivo fluorescence images were acquired using a Carestream In-Vivo MS FX Pro (Bruker) imaging station using an excitation filter of 630 nm and an emission filter of 700 nm for Cy5 (190 mm field-of-view, f-stop 2.8, 30 s acquisition time) and were coregistered with an X-ray image (0.2 mm aluminum filter, 1.2 s acquisition time). All images were processed using ImageJ (National Institutes of Health). A subset of mice administered targeted nanomedicines were injected with 100 μL of tomato lectin vascular marker (DyLight 594 Lycopersicon esculentum lectin, DL01177, Vector Laboratories, CA) 1 min prior to euthanasia at the 48 h time point. After ex vivo images were acquired, tumors were paraformaldehyde fixed (4% (w/v) in cold phosphate buffered saline) overnight before being embedded in paraffin, sectioned, and 4′,6-diamidino-2-phenylindole (DAPI) stained. Cy5, DAPI, and vascular marker localization was then imaged using confocal microscopy (Zeiss LSM 710).

Synthesis and Structure Confirmation of Hyperbranched Polymers. The hyperbranched polymer was successfully prepared using the well-established RAFT polymerization method utilized extensively in our group.32,34−36 In this study the monomers used in the feed were PEGMA, a pH sensitive linker (N-(tert-butoxycarbonyl)-N′-(6methacrylamidohexanoyl)hydrazine; TBMC), Cy5-MA, and EGDMA as a branching agent. The molecular weight of each arm of the polymer was evaluated by 1H NMR (Figure S2), where the integration of distinct peaks within each monomer showed a ratio of PEGMA:TBMC:RAFT = 16:4:1. Thus, it was estimated that the molecular weight of each arm of HBP-1 was about 10.49 kDa, with roughly 20 mol % incorporation of TBMC. The absolute molecular weight of the HBP was measured using GPC-MALLS to give an indication of the overall size of the branched polymer. The number of arms that make up each HBP was determined to be three, and was calculated by comparing the absolute molecular weight (Mn(SEC‑MALL)) and the arm molecular weight (Mn(NMR)). As a means of validating the molecular structure, UV−vis spectroscopy was also used to estimate the number of Cy5-MA units in each polymer (ε = 177207 M−1 cm−1, λ = 647 nm, water). On average, every ten polymers contained one Cy5 molecule, this being of sufficient concentration for in vitro and in vivo fluorescence imaging studies. HBP-2 was synthesized by deprotecting the acid groups of the methcrylic acid units. Using 10% TFA/DCM at 40 °C for 3 h, t-boc groups on the TBMC repeat units were removed from HBP-1. 1H NMR showed that following this treatment the peak characteristic of the methyls in the t-boc protecting group at about 1.5 ppm had completely disappeared following this treatment, confirming successful generation of the hydrazide methacrylamide unit (Figure S3; inset shows disappearance of peak b). Importantly, UV−vis confirmed that under these conditions there was not significant degradation of the Cy5 moieties (∼80% Cy5 groups remained). The peptide aptamer modified with an N-terminal (5azidopentanoic acid) azide group was reacted with the alkyneterminated HBP (HBP-2) via copper-catalyzed azide−alkyne cycloaddition. Following reaction, 1H NMR clearly shows the presence of a new peak at 5.11 ppm, which is the characteristic peak for the methylene group adjacent to the triazole. The simultaneous disappearance of the proton resonance at 4.5 ppm that was attributed to the methylene protons adjacent to the alkyne group in the starting polymer (HBP-2) confirms that the click reaction proceeded to high conversion and indicates that the aptamer is attached to each arm of the HBP (HBP-3) (Figure S4). Attachment of DOX to the HBP precursors (HBP-4 and HBP-5) was achieved by the reaction of hydrazide groups with the ketone present in DOX at the C13 position, as described in previous publications.37 1H NMR provided evidence that DOX had attached to the HBP through the appearance of aromatic peaks at 7−8 ppm. Through comparison of the integrals of signals attributed to the DOX and the polymer end-groups, it was found that there were approximately six DOX molecules per polymer. This was further verified by UV−vis spectroscopy, which also suggested that there were approximately 6 DOX molecules per polymer (Figure S7). In Vitro DOX Release. The in vitro doxorubicin release profile was determined using a sodium phosphate aqueous solution that mimicked the pH of physiological parameters that might be encountered in animals. The release of DOX from

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RESULTS AND DISCUSSION The main purpose of this study was to develop a targeted, dualtherapeutic polymeric nanomedicine for both therapeutic and diagnostic purposes and to undertake preliminary studies into targeting efficacy. An aptamer targeted hyperbranched polymer was successfully synthesized which provided both targeting and therapeutic functions by using an 8-mer peptide aptamer as the ligand against Hsp70. In addition, the anticancer therapeutic DOX was also bound to the polymeric carrier utilizing a degradable hydrazone bond, enabling pH-controlled release under mildly acidic conditions that mimics the environment in endosomes/lysosomes of tumor cells. The general synthetic pathway for synthesis of this polymeric platform is described in Scheme 1, and Table 1 shows the physicochemical properties of the nanomedicine. Table 1. Physical and Chemical Properties of the Hyperbranched Polymer Scaffold, HBP-1 Mn (kDa)

HBP-1 a

monomer conversion

SECMALLS

H NMR

no. of branch points

Dh (nm)

no. of Cy5 dyesb/ polymer

>95%

34

10.5

3.2

6.7 ± 1.6

0.1

1

a

Determined by DLS. bDetermined by UV−vis spectroscopy. E

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will be phagocytosed by cells owing to their settling onto cell surfaces in 2D culture. It is also recognized that the expression of Hsp70 is strongly increased in vivo when cells undergo periods of stress, while in vitro the microenvironment is less stress-inducible, which may lead to low expression levels of Hsp70 protein with subsequently lower observed targeting.41 IC50 values for both cell lines were also measured and summarized in Table 2, and it was noted that the IC50 values of free DOX were lower than those of HBP-4 and HBP-5 nanocarriers in both cancer cell lines. This is a common phenomenon in many nanopolymer drug delivery systems42 and may reflect a different route or different kinetics for the uptake of HBP-4 and HBP-5 compared with free DOX. In Vitro Cellular Binding/Uptake Study. Flow cytometry analysis was performed to compare binding of HBP-2, HBP-3, HBP-4, and HBP-5 using MDA-MB-468 breast cancer cell line. As shown in Table 3, both HBP-3 and HBP-5 have enhanced binding compared to HBP-2 and HBP-4. This indicated that the aptamer can enhance the interaction of the polymeric carrier with the cells by targeting Hsp70 and that both HBP-3 and HBP-5 were most likely transported within cells by an aptamer-receptor-mediated endocytosis process. In addition, a comparison of HBP-2 and HBP-4 shows that aptamer targeting helps increase the cellular association of HPBs. To further validate that the cellular association is via a receptor-mediated mechanism, a competitive binding assay was performed. Table 3 shows that when the cells are incubated with a mixture of free aptamer and HBP-3, the cellular association is significantly decreased compared to that of HPB-3 alone, suggesting that the receptor binding sites were being efficiently blocked by the free aptamer. Live cell confocal microscopy was used to investigate the internalization behavior of the materials by the MDA-MB-468 cell line. Figure 3 compares the distribution of free DOX, the untargeted polymer (HBP-2), and the Hsp70 targeted polymer (HBP-3) in live cells following incubation for 2 h. All samples displayed uptake into the cells with free DOX located in membranes and small punctate structures, in addition to the nucleus (Figure 3A). The free untargeted polymer displayed diffuse cytoplasmic staining with aggregation in the inner cytoplasm (Figure 3B). In contrast, the Hsp70 targeted polymer systems were predominantly restricted to large vesicular structures (see Figure 3C). These observations suggest that both DOX and free polymer enter the cellular milieu via nonspecific means such as hydrophobic membrane interactions and macropinocytosis. While further study is required, the aggregation of the targeted HBP into large vesicles supports the hypothesis that the material has gained access to the endolysosomal system through the predicted cell-surface marker mediated process. Figures 4A and 4B compare the DOX and Cy5 channels at 2 h postexposure of HBP-5 to MDA-MB-468 cells. Significant staining of the plasma membrane and a high level of cytoplasmic staining were observed, with the accumulation of both Cy5 and DOX fluorescence toward the nucleus with an appreciable amount of vesicular staining (see Figure 4). While Cy5 and DOX fluorescence were predominantly colocalized (see Figure 4C), there is evidence of DOX staining at the plasma membrane and there are vesicles present which appear to be predominantly laden with Cy5. This suggests that chemotherapeutic release has begun within this time period and the DOX has distributed into the nucleus. The change in fluorescence distribution for both fluorescent components is

HBP-4 was investigated using reverse phase high performance liquid chromatography (HPLC) with fluorescent detection of doxorubicin (excitation at 480 nm, and emission at 560 nm). Details of the procedure for investigating the amount of DOX released from the polymer conjugate after incubation in both pH = 5 and pH = 7.4 sodium phosphate buffer at 37 °C are shown in Figure S6. It was shown that the DOX attached via the hydrazone bond was relatively stable under conditions that mimic blood plasma (pH 7.4; 37 °C), where less than 10% of DOX was released from the polymer over 48 h. In contrast, release of DOX at pH 5.0 was quite efficient, with 50% of the drug being released after approximately 12 h and over 80% released after 48 h. This pH was chosen to represent the acidic environment that might be encountered upon internalization into endosomes/lysosomes in tumor cells. The delivery system performed to a high standard when compared to similar studies of release kinetics of DOX from pH sensitive nanoparticles that use a hydrazone bond.37−39 In general, as shown in Figure 1,

Figure 1. Release profile of DOX from HBP-4 in a sodium phosphate aqueous solution at pH 5.0 (red) and pH 7.4 (blue) at 37 °C.

this pH responsive HBP was shown to be an efficient prodrug, because it was relatively stable in conditions that are typical of circulating blood, with good release of DOX when exposed to conditions representing those that are typical of the lysosome. In Vitro Cell Viability Study. The in vitro cytotoxicity of HBP-2, HBP-3, HBP-4, HBP-5, and free DOX against breast cancer cells MDA-MB-231 and MDA-MB-468 was evaluated using MTS assay, with an incubation time of 48 h.40 The cell viability of MDA-MB-231 (Figure 2A,B) and MDA-MB-468 (Figure 2C,D) in the presence of HBP-2, HBP-3, HBP-4, HBP-5, and free DOX at various concentrations is presented. From this data, it can be concluded that there is no significant cytotoxicity observed for the empty vehicle (HBP-2) on either MDA-MB-231 cell line or MDA-MB-468 cell line. HBP-3 was also well tolerated by both cell lines, confirming that this aptamer−polymer conjugate is not considered cytotoxic; this is in agreement with literature reports that suggest that the aptamer is not cytotoxic on its own.31 Both DOX nanoconjugates (HBP-4 and HBP-5) and free DOX significantly decreased viability in both cell lines. Being a small molecule, free DOX diffuses into cells quickly and therefore showed greater toxicity toward both cell lines. However, as shown in both Figures 2B and 2D, the cell viability in the presence of HBP-5 and HBP-4 does not exhibit significantly different results between the targeted and untargeted species. This could be due to a number of reasons, but most likely to the fact that the MTS assay was conducted following 48 h incubation; during this time it is well-established that most nanomaterials F

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Figure 2. Cell viabilities determined by the MTS assay of two breast cancer cell lines treated with different formulations of the nanocarrier at a range of concentrations. MDA-MB-231 cells were treated with (A) HBP-2 and HBP-3 and (B) DOX-containing formulations HBP-4 and HBP-5 and free DOX. MDA-MB-468 cells were treated with HBP-2 and HBP-3 (C) and DOX-containing formulations HBP-4 and HBP-5 and free DOX (D).

Table 2. IC50a of Free DOX, as Well as Targeted and Untargeted Polymers Conjugated with DOX, on Breast Cancer Cells

At 4 h, clear divergence between the fluorescence signals can be seen, with DOX showing its highest fluorescence in the nucleoplasm and the Cy5 labeled HBP remaining prevalent in vesicular structures predominantly toward proximal regions of the cell (see Figure 5; Figures S8 and S9). The uncoupling of the two fluorophores taken in conjunction with the vesicular preference of HBP-3 indicates that the targeting-ligand directs the material along the endolysosomal pathway, facilitating the cleavage of the DOX from the polymer. This is further evidenced by DOX switching from a similar cytoplasmic motif as the HBP to the reported moiety. The behavioral changes between 2 and 4 h were confirmed via the ROI analysis (see Figure 5D), which identifies a 4-fold increase in DOX intensity within nuclear regions and a decrease in all of the cytoplasmic elements examined. Alternatively, Cy5 distribution remains relatively unchanged particularly within vesicular structures. It is of note that numerous cells exhibited bright Cy5-positive vesicles at the plasma membrane which were not present in the DOX channel; this could be a potential sign of exocytosis after cleavage and drug release but requires further investigation to confirm this hypothesis. In Vivo Targeting Study. In vivo optical imaging was used to show the enhanced accumulation of the aptamer targeting vector in BALB/c nude mice bearing MDA-MA-468 xenograft tumors in the mammary fat pad. Images 48 h postinjection (Figure 6) clearly showed higher tumor accumulation of the targeted HBP-5 when compared to untargeted HBP-4. Further confirmation of this higher accumulation can be seen in the relative intensity from the various organs following excision from mice at 24 and 48 h (Figure 7; Figure S11 and Table S1). One important observation from Figure 6B is that, after 24 and

IC50 (μg/mL)

a

cell lines

DOX

HBP-4

HBP-5

MDA-MB-231 MDA-MB-468

0.09 (±0.03) 0.08 (±0.01)

0.54 (±0.06) 0.71 (±0.05)

0.43 (±0.02) 0.60 (±0.06)

Inhibitory concentration required to produce 50% cell death.

Table 3. FACS Analysis of the Cellular Association of the Different Formulations of HBP with MDA-MB-468 Breast Cancer Cellsa control 0.4

HBP-2

HBP-3

HBP-4

HBP-5

25.2 (±0.5)

97.2 (±0.3)

47.8 (±0.3)

96.9 (±0.6)

HBP-3 and aptamer 23.4 (±0.9)

a Percentage of cells (MDA-MB-468) showing cyanine-5 fluorescence from a sample population of 10000 cells.

confirmed by semiquantitative ROI analysis (see Figures 4D and 4E). Importantly, the addition of the targeting ligand significantly increases the presence of the polymer within vesicles and the nuclear environment (Figure 4D). Figure 5 shows the cellular staining of live MDA-MB-468 cells following incubation with HBP-5 for 4 h, where the nuclear staining with DOX is clearly defined. This shifts from a predominantly nucleoplasmic localization to possessing a cytoplasmic presence, indicating that DOX is being delivered via its attachment to the HBP and not freely diffusing. G

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Figure 3. Confocal fluorescence micrographs of live MDA-MB-468 cells 2 h postexposure to free DOX (A), untargeted HBP-2 (B), and Hsp70 targeted HBP-3 (C). The nuclei of example cells have been marked (n); examples of large vesicular structures containing HBP-3 are marked with arrows. Scalebar denotes 5 μm.

Figure 4. Confocal fluorescence micrographs of live MDA-MB-468 cells 2 h postexposure to HBP-5 (A−C): DOX fluorescence (A), Cy5 signal (B), fluorescence channels merged (C); colocalization of DOX and Cy5 appears white; the nuclei of selected cells have been marked (n). Cy5-labeled vesicles are marked with arrowheads, and DOX staining of the plasma membrane is indicated with an arrow. Scale bars as labeled. ROI analysis comparing normalized fluorescence intensity distribution of the cellular level images between the Cy5 channels of HBP-2 and HBP-3 (D) and free DOX and HBP-5 (E). Scale bar in images denotes 5 μm.

determination of the nanomedicine distribution within the tumor on a cellular scale. Microscopy of the tumor slice (Figure 8) allowed visualization of both the nanomedicine and drug distribution relative to tumor-tissue cells (nuclei stained blue) and blood vessels (magenta). This showed that the polymeric carrier remained primarily at the blood vessel boundaries, while DOX was observed to be distributed throughout the various cells within the tumor tissue. These results suggested that, following nanomedicine accumulation at the tumor site, DOX was released from the polymer, presumably due to the acidic tumor microenvironment either inside or outside cells, and

48 h postinjection, the tumor accumulation of Cy5 from targeted HBP-5 was increasing while the untargeted polymer (HBP-4) signal decreased. These results suggested that HBP-5 not only has higher accumulation in the tumor tissue but also has longer retention owing to the ligand−receptor interaction of the aptamer targeting vector. Another important observation from these studies is that the polymeric nanomedicine was being cleared predominantly via the liver. Intratumor Distribution of Nanomedicine. Following whole organ fluorescence imaging, the tumors were fixed, sectioned, and imaged using confocal microscopy to allow H

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Figure 5. Confocal fluorescence micrographs showing DOX (A) and Cy5 (B) distribution at 4 h, as well as DOX and Cy5 channels overlaid (C); examples of DOX staining of the plasma membrane and Cy5-positive vesicles marked with an arrow and arrowheads, respectively. Comparison of relative fluorescence intensity of DOX and Cy5 within the various compartments of live MDA-MB-468 cells exposed to HBP-5 for 2 and 4 h (D). Scalebar in images denotes 5 μm.

then proceeded to enter the nucleus and cause cell apoptosis. Meanwhile, most of the polymer nanocarrier remained in the tissue close to blood vessels. In general, this might imply that, while the polymer acts as an efficient carrier for the drug to reach the tumor tissue, diffusion of the drug is likely to occur in the free form through the tissue mass providing access to the other cells within the tissue.43,44

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CONCLUSIONS A targeted theranostic hyperbranched polymer was successfully synthesized and characterized. The Hsp70 targeted aptamer incorporated onto the polymer can provide significantly enhanced accumulation within xenograft tumors compared to the untargeted analogue. In addition, controlled release of DOX in vitro was achieved via the pH sensitive hydrazone bond, which is relatively stable at physiological conditions (pH 7.4), but can effectively be cleaved under mildly acidic conditions (pH 5) which mimic those found in endosomal and lysosomal environments. HBP-5 had a greater cellular uptake when compared with HBP-4 due to the specificity of the 8-mer peptide aptamer toward Hsp70. In live MDA-MB-468 cells, targeted HBP-5 was shown to accumulate in vesicles and therapeutic release was observed. Finally, the in vivo study suggested that HBP-5 not only had higher accumulation in the tumor tissue but also had longer retention at the site of the tumor presumably because of the ligand−receptor interaction.

Figure 6. (A) In vivo fluorescence images of mice bearing xenograft solid tumor (MDA-MB-468 cells) at 48 h postinjection of HBP-4 and HBP-5. (B) ROI analysis of the ex vivo solid tumor at both 24 and 48 h postinjection of HBP-4, HBP-5 (n = 3), and saline as control.

I

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Figure 7. Ex vivo biodistribution of different HBPs in mice. Intensities were measured by fluorescence imaging in each organ based on Cy5 signal. (A) Normalized fluorescence intensity of untargeted HBP-4 (n = 3) at 24 and 48 h. (B) Normalized fluorescence intensity of targeted HBP-5 (n = 3) at 24 and 48 h.

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ACKNOWLEDGMENTS The researchers would like to thank Dr. Christopher Howard (Australian Institute for Bioengineering and Nanotechnology, Australia) for the cancer cells used in this study. This work was performed in part at the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano- and microfabrication facilities for Australia’s researchers. This research utilized equipment provided by the QLD node of the National Biologics Facility (www. nationalbiologicsfacility.com), an initiative of the Australian Government being conducted as part of the NCRIS National Research Infrastructure for Australia. We acknowledge funding from the National Health and Medical Research Council (APP1099321, K.J.T.), the Australian Research Council (FT110100284 (K.J.T.), DP140100951 (K.J.T.)), and the Australian Commonwealth Government International Postgraduate Research Scholarship (Y.Z.). We also acknowledge the National Breast Cancer Foundation for funding (K.J.T., N.L.F.). This research was conducted and funded by the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036).

Figure 8. Confocal images of an ex vivo tumor slice (nucleus, blue; DOX, green; polymer, red; and blood vessel, magenta) at different magnification: (A) scale bar denotes 50 μm; (B) scale bar denotes 10 μm; (C) scale bar denotes 5 μm.

Overall, all these features indicate that the reported HBP platform is a promising theranostic platform for breast cancer therapy.

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ASSOCIATED CONTENT

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00560. NMR and UV−vis spectra and ex vivo flow cytometric analyses (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Zachary H. Houston: 0000-0001-9738-4917 Adrian V. Fuchs: 0000-0002-8112-0527 Idriss Blakey: 0000-0003-2389-6156 Kristofer J. Thurecht: 0000-0002-4100-3131 Notes

The authors declare no competing financial interest. J

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