Sugar Concentration and Arrangement on the Surface of

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Cite This: Biomacromolecules XXXX, XXX, XXX−XXX

Sugar Concentration and Arrangement on the Surface of Glycopolymer Micelles Affect the Interaction with Cancer Cells Mingxia Lu,† Yee Yee Khine,† Fan Chen,† Cheng Cao,†,‡ Christopher J. Garvey,‡ Hongxu Lu,† and Martina H. Stenzel*,† †

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Centre for Advanced Macromolecular Design (CAMD), School of Chemistry, University of New South Wales, Sydney NSW 2052, Australia ‡ Australia Nuclear Science and Technology Organisation, Lucas Heights, NSW 2234, Australia S Supporting Information *

ABSTRACT: Glycopolymer-coated nanoparticles have attracted significant interest over the past few years, because of their selective interaction with carbohydrate receptors found on the surface of cells. While the type of carbohydrate determines the strength of the ligand−receptor interaction, the presentation of the sugar can be highly influential as the carbohydrate needs to be accessible in order to display good binding. To shine more light on the relationship between nanoparticle structure and cell uptake, we have designed several micelles based on fructose containing block copolymers, which are selective to GLUT5 receptors found on breast cancer cell lines. The polymers were based on poly-D,L-lactide (PLA), poly(2hydroxyethyl) acrylate (PHEA), and poly(1-O-acryloyl-β-D-fructopyranose) (P[1-O-AFru]). A set of six micelles was synthesized based on four fructose containing micelles (PLA242-b-P[1-OAFru]41, PLA242-b-P[1-O-AFru]179, PLA242-b-P[1-O-AFru46-c-HEA214], PLA242-b-PHEA280-b-P[1-O-AFru]41) and two neutral controls (PLA247-b-PHEA53 and PLA247-b-PHEA166). SAXS analysis revealed that longer hydrophilic polymers led to lower aggregation numbers and larger hydrophilic shells, suggesting more glycopolymer mobility. Cellular uptake studies via flow cytometry and confocal laser scanning microscopy (CLSM) confirmed that the micelles based on PLA242-b-P[1-O-AFru]179 show, by far, the highest uptake by MCF-7 and MDA-MB-231 breast cancer cell lines while the uptake of all micelles by RAW264.7 cell is negligible. The same micelle displayed was far superior in penetrating MCF-7 cancer spheroids (threedimensional (3D) model). Taking the physicochemical characterization obtained by SAXS and the in vitro results together, it could be concluded that the glycopolymer chains on the surface of micelle must display high mobility. Moreover, a high density of fructose was found to be necessary to achieve good biological activity as lowering the epitope density led immediately to lower cellular uptake. This work showed that it is crucial to understand the micelle structure in order to maximize the biological activity of glycopolymer micelles.

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INTRODUCTION Glycocalyx are crucial components in biological functions. They are involved in cell adhesion, cell−cell recognition, and proliferation. Inspired by the importance of carbohydrates in biological processes, glycopolymers1−3 have been widely employed in the design of nanoparticles, because of the “cluster glycoside effect”, which is a multivalent effect that established the strong binding between glycopolymers and their receptors.4 The ability of glycopolymers to interact with specific lectins has been subject to intense investigations for many years now.1−3,5 It is known that the binding effect is dependent on the molecular weight,6,7 the epitope density,7 the polymer architecture,8−10 the arrangement of the sugar either in linear or dendritic structure,11 and even the length of spacer groups between polymer backbone and the pendant sugar.10,12 Most of these studies have been performed using free polymer chains © XXXX American Chemical Society

in solution. However, it must be considered that glycopolymers that are part of a hierarchical assembly, such as micelles, have only limited conformational freedom, similar to glycopolymers on surfaces. Here, the polymer movement is restricted, depending on the polymer density, taking on either a “mushroom” state at low chain density or a “brush” state.13 The interaction of glycopolymers with lectins grafted onto surfaces has been subject to intense studies.14−16 A detailed investigation on the interaction between galactose-based glycopolymer brushes, grafted onto gold surfaces, and Ricinus communis agglutinin (RCA120) reported better binding with Special Issue: Biomacromolecules BPC Received: September 19, 2018 Revised: November 27, 2018

A

DOI: 10.1021/acs.biomac.8b01406 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules Scheme 1. Block Copolymers Synthesized for This Study

high-density brushes.17 Similar correlations were found in other systems based on glucose chains that bind to ConA, where high grafting densities were found to be advantageous.18 Thicker brushes, which are obtained with longer polymers, can enhance binding until a critical steric hindrance has been reached that prevents lectin penetration into the brush.17,19 Meanwhile, it showed that it is not necessary to have a high concentration of carbohydrates on the surface.20 Lectin binding increases initially with increasing carbohydrate concentration, until further sugar addition results in reduced binding.17 The studies on lectin binding are usually performed in buffer solution with glycopolymers and lectin only. However, one must remember that, in living systems, the glycopolymer may not interact with the lectin alone, but adsorption of other proteins found in the blood may be prevalent.21−24 Despite the potentially competing nonspecific adsorption of proteins found in biological systems, glycopolymer were found to display high binding to surface receptors on cells in a biological environment. For example, GLUT5 overexpressing breast cancer cells can distinguish between different sugar-based micelles displaying a high affinity to fructose-coated nanoparticles.25,26 Polymeric micelles with fructose pendants showed high selectivity to breast cancer cells while they display low uptake by healthy cells,27,28 thanks to the overexpression of GLUT5 transporters in breast cancer tissue.29,30 The extracellular asialoglycoprotein receptors (ASGP-R) on liver cancer cell HepG2 are capable of detecting different spatial orientations of galactose on the surface of nanoparticles.31 Polymers with large amount of galactose were observed to display efficient transfection of hepatocytes,32 while displaying low uptake by cells with low receptor expression.33 Galactose-based nanoparticles also display high uptake by HeLa cells with the amount of endocytosis being strongly dependent on the polymer architecture of the glycopolymer-based nanoparticle.34 Also, macrophages appear to have a high affinity to mannose-based nanoparticles.35−37 Even single carbohydrate endfunctionalities on the polymers are sufficient, as demonstrated in the delivery of galactose decorated polymersomes to HepG2 cells38 or the delivery of glucose-functionalized micelles to the blood-brain barrier.39 The focus in the current glyconanoparticles literature is on the relationship between the type of carbohydrate and the cellular uptake by various cell lines. There is some interest in

the effect of the nanoparticle shape,28,35,36,40,41 but there are limited reports that look at the presentation of the glycopolymer chains on the surface, such as the sugar density42 and its correlation to cell affinity. The aim of this work is to understand how the length of the glycopolymer in the shell of the micelle, but also the concentration and arrangement of carbohydrateshere, GLUT5 targeting fructosewill affect the activity. Therefore, six block copolymers were designed: PLA-b-PHEA with two different HEA chain lengths, PLA-b-PFru with two different fructose chain lengths, a block copolymer with statistical arrangement of HEA and Fru, PLA-b-P(HEA-co-Fru), and a triblock copolymer (PLA-b-PHEA-b-PFru) (Scheme 1). The self-assembled polymeric micelles with PLA core were characterized by DLS and SAXS to unveil information on the structure of the polymer in the shell. The question that will be answered include whether there is a difference in the length of the fructose chain on the surface, and if the epitope density will play a role or if small amounts of fructose are sufficient to achieve good cellular uptake.

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MATERIALS AND METHODS

Chemicals. All chemicals were reagent-grade and used as received, unless otherwise specified. N,N-dimethylacetamide (DMAc; 99.9%, Sigma−Aldrich), fluorescein O-methacrylate (97%, Sigma−Aldrich), trifluoroacetic acid (TFA; 99%, Sigma−Aldrich), deuterated dimethylsulfoxide-d6 (DMSO-d6; Cambridge Isotope Laboratories), diethyl ether (99%, Ajax Finechem), dimethylsulfoxide (DMSO, Chem-supply Pty Ltd.), methanol (Merck Pty Limited) were used without any further purification. 2-Hydroxyethyl acrylate (HEA; 96%, Aldrich) was destabilized by passing it over a column of basic alumina. 2,2-Azobis(isobutyronitrile) (AIBN; 98%, Fluka) was purified by recrystallization from methanol. 1-O-acryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose (1-O-AiPrFru) fructose monomer was prepared according to the previous method.30 Poly-D,L-lactide (PLA) MacroRAFT agent was synthesized according to the procedure described in the previous work.30 Milli-Q water was produced internally and had a resistivity of 18.2 mΩ cm−1. Synthesis. Synthesis of PLA−PHEA Block Copolymer. For the short HEA chain polymer synthesis: HEA (90 mg, 9.7 × 10−1 mol L−1), PLA MacroRAFT (138 mg, 9.7 × 10−3 mol L−1), fluorescein Omethacrylate (0.9 mg, 2.8 × 10−3 mol L−1) and AIBN (0.13 mg, 9.7 × 10−4 mol L−1) as initiator were dissolved in DMAc (0.8 mL), to give a ratio of [HEA]/[fluorescein O-methacrylate]/[MacroRAFT]/ [AIBN] = 100:0.3:1:0.1. B


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Biomacromolecules

(BHT), 0.03% w/v of LiBr) with a flow rate of 1 mL min−1 was used for analyses. The injection volume was 50 μL. The samples were prepared by dissolving 2−3 mg mL−1 of the analyte in DMAc, followed by filtration through a 0.45 μm filter. The unit was calibrated using commercially available linear poly(methyl methacrylate) standards (0.5−1000 kDa, Polymer Laboratories). Chromatograms were processed using Cirrus 2.0 software (Polymer Laboratories). Dynamic Light Scattering (DLS). The average hydrodynamic diameter of micelles in aqueous solution were determined using a Malvern Zetasier Nano ZS instrument with a 4 mV He−Ne laser operating at λ = 632 nm and noninvasive backscatter detection at 173°. Measurements were performed in a disposable cuvette with a solution of 1 mg mL−1 polymer in distilled water at 25 °C. Transmission Electron Microscopy (TEM). The TEM micrographs were obtained using a JEOL1400 TEM system, consisting of a beam voltage of 100 kV and a Gatan CCD facilitating the acquisition of digital images. Samples were prepared by casting a 0.5 mg mL−1 polymeric micelle solution onto a copper grid. The grids were airdried and then negatively stained with uranylacetate (UA) for 1 min, followed by being air-dried overnight. Fluorescence Spectroscopy. Fluorescence spectra of samples were obtained on Cary Ecliose Fluorescence Spectrophotometer (Aglient Technologies). The excitation and emission wavelength were 491 and 512 nm, respectively. Small-Angle X-ray Scattering (SAXS). SAXS experiments were performed at the Australian Synchrotron on the small/wide-angle Xray scattering beamline using X-rays with a wavelength (λ) of 1.127 Å. Isotropic scattering patterns were collected on a Pilatus 1 M detector with an active area of 981 × 1043 pixels of 170 m2 each with a 2.7 m sample-to-detector distance. The samples were placed in a 96-well plate solution autoloader from where samples were taken automatically and the SAXS was measured in a consistent position in a quartz capillary. The data were converted to the familiar absolutely scaled scattering cross-section as a function of the scattering vector, q, I(q). The magnitude of scattering vector q is defined by

For the long HEA chain polymer synthesis: HEA (180 mg, 9.7 × 10 mol L−1), PLA MacroRAFT (69 mg, 2.4 × 10−3 mol L−1), fluorescein O-methacrylate (1.8 mg, 2.8 × 10−3 mol L−1) and AIBN (0.06 mg, 2.4 × 10−4 mol L−1) were combined in DMAc (1.6 mL), to give a ratio of [HEA]/[fluorescein O-methacrylate]/[MacroRAFT]/ [AIBN] = 400:1.2:1:0.1. The solutions were deoxygenated by purging with nitrogen for 45 min, and then polymerized at 70 °C for 2 and 3.5 h respectively. The final product was precipitated into diethyl ether and dried under vacuum. The obtained polymer was confirmed by 1H NMR. Synthesis of PLA-P(1-O-AiPrFru) Block Copolymer. For the short fructose chain polymer: 1-O-AiPrFru (100 mg, 9.8 × 10−1 mol L−1), PLA MacroRAFT (111 mg, 2.0 × 10−2 mol L−1), fluorescein Omethacrylate (1 mg, 7.7 × 10−3 mol L−1) and AIBN (0.5 mg, 9.7 × 10−3 mol L−1) were dissolved in DMAc (0.324 mL), to give a ratio of [1-O-AiPrFru]/[fluorescein O-methacrylate]/[MacroRAFT]/[AIBN] = 50:0.4:1:0.5. For the long fructose chain polymer: 1-O-AiPrFru (100 mg, 9.8 × 10−1 mol L−1), PLA MacroRAFT (28 mg, 4.9 × 10−3 mol L−1), fluorescein O-methacrylate (1 mg, 7.7 × 10−3 mol L−1) and AIBN (0.13 mg, 2.4 × 10−3 mol L−1) were dissolved in DMAc (0.324 mL), to give a ratio of [1-O-AiPrFru]/[fluorescein O-methacrylate]/ [MacroRAFT]/[AIBN] = 200:1.6:1:0.5. The solution was deoxygenated by purging with nitrogen for 45 min, and then polymerized at 70 °C for 3.5 h. The polymer was precipitated into cold methanol and dried under vacuum. The obtained polymer was confirmed by 1H NMR. Synthesis of PLA- [PHEA-co-P(1-O-AiPrFru)] Diblock Terpolymer. 1-O-AiPrFru (30 mg, 2.1 × 10−1 mol L−1), HEA (55 mg, 1.06 mol L−1), PLA MacroRAFT (33 mg, 4.2 × 10−3 mol L−1), fluorescein Omethacrylate (0.85 mg, 4.2 × 10−3 mol L−1) and AIBN (0.16 mg, 2.1 × 10−3 mol L−1) as initiator were dissolved in DMAc (0.45 mL), to give a ratio of [1-O-AiPrFru] [HEA]/[fluorescein O-methacrylate]/ [MacroRAFT]/[AIBN] = 50:250:1:1:0.5. The solution was deoxygenated by purging with nitrogen for 45 min, and then polymerized at 70 °C for 3.25 h. The polymer was precipitated into diethyl ether and dried under vacuum. The obtained polymer was confirmed by 1H NMR. Synthesis of PLA- b-PHEA-b-P(1-O-AiPrFru) ABC Triblock Terpolymer. PLA-P(1-O-AiPrFru) (60 mg, 2.3 × 10−3 mol L−1), HEA (65 mg, 9.3× 10−1 mol L−1), fluorescein O-methacrylate (0.65 mg, 2.7 × 10−3 mol L−1) and AIBN (0.02 mg, 2.3 × 10−4 mol L−1) as initiator were dissolved in DMAc (0.6 mL), to give a ratio of [HEA]/[ fluorescein O-methacrylate]/[PLA-P(1-O-AiPrFru)]/[AIBN] = 400:1:1:0.1. The solution was deoxygenated by purging with nitrogen for 45 min, and then polymerized at 70 °C for 3.25 h. The polymer was precipitated into diethyl ether and dried under vacuum. The obtained polymer was confirmed by 1H NMR. Deprotection of Fructose-Containing Polymers. The fructosecontaining polymers (100 mg) was mixed with 2 mL of TFA/H2O (5:1 v/v) at room temperature and stirred for 40 min. The solution was dialyzed against Milli-Q water (MWCO = 3500 g mol−1) for 2 days and freeze-dried to give the final products. Micellization of Copolymer. Ten milligrams (10 mg) of the copolymer dissolved in a mixture (0.2 mL) of DMSO. The mixture then was dropwise added to 2.5 mL of Milli-Q water. The solution was subsequently dialyzed against Milli-Q water to remove the organic solvent and diluted to 5 mL. (MWCO = 3500 g mol−1). Nuclear Magnetic Resonance (NMR) Spectrometry. All the NMR spectra were obtained by using a Bruker Avance III 300 (1H, 300.17 MHz), 5 mm BBFO probe. Samples were analyzed in the solvents DMSO-d6. All chemical shifts are stated in ppm (δ), relative to tetramethylsilane (δ = 0 ppm). Size Exclusion Chromatography (SEC). Molecular weight and distributions were determined by SEC using a Shimadzu modular system containing a DGU-12A degasser, a LC-10AT pump, a SIL10AD automatic injector, a CTO-10A column oven, and a RID-10A refractive index detector. A 50 × 7.8 mm guard column and four 300 × 7.8 mm linear columns (500, 103 104, and 105 Å pore size, 5 μm particle size) were used for the analyses. N,N-Dimethylacetamide (DMAc, HPLC grade, 0.05% w/v of 2,6-dibutyl-4-methylphenol −1

q=

4π iθ y sinjjj zzz λ k2{

where θ is the scattering angle. A background subtraction was performed using a solvent filled capillary scaled by the transmission of sample and solvent capillaries. The SAXS data reduction: conversion of raw pixel counts, measurement geometry and X-ray wavelength into a radial average; background subtraction; and the normalization to the scattered intensity of water as a secondary intensity standard was performed by the beamline program Scatterbrain. Data were analyzed by modeling the measured I(q) with model structure, where the structural parameters were optimized using the program SASview.43 The starting point for the model was a spherical core−shell particle, where the core consisted of PLA and the shell a mixture of the water and polyfructose with defined radii. This compositional information was input into the model, in terms of the electron density or scattering length density.44 In Vitro Cell Culture. Human breast carcinoma MDA-MB-231 cells, human breast carcinoma MCF-7 cells, and mouse macrophages RAW264.7 were cultured in T25 cell culture flasks with 5% CO2 at 37 °C. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% glutamax, and 0.01% plasmocin. After reaching 70% confluence, the cells were washed with phosphate buffered saline (PBS) and collected after trypsin/ethylenediaminetetraacetic acid (EDTA) treatment. Cytotoxicity Assay. The cytotoxicity of micelles against MCF-7 cells was measured by WST-1 assay. The cells were seeded at a density of 4 × 103 cells per well in 96-well cell culture plates and incubated at 37 °C in a 5% CO2 environment for 1 d prior to micelle treatment. Micelle solutions (1 mg mL−1) were sterilized by UV irradiation for 15 min in a biosafety cabinet, then serially diluted (2× dilution) with sterile Milli-Q water. The cell culture medium was C


Article

Biomacromolecules Table 1. Summary of Polymers Prepared No. 1 2 3 4 5 6

polymer PLA247-b-PHEA53 PLA247-b-PHEA166 PLA242-b-P[1-O-AiPrFru]41 PLA242-b-P[1-O-AiPrFru]179 PLA242-b-P[1-O-AiPrFru46-cHEA214] PLA242-b-PHEA280-b-P[1-OAiPrFru]41

conversion of [1-O-AiPrFru] (1H NMR)

conversion of [HEA] (1H NMR)

Mn, NMR (g mol−1)

Mn, sec (g mol−1)

Đ

82 89.5 92

53 41.5 − − 85.6

23 900 37000 30 300 73 600 56 700

33 000 41 000 16 000 13 000 27 000

1.23 1.26 1.13 1.24 1.24

82

70

62 800

30 000

1.21

with (−)-epicatechin-gallate (100 μM) followed by an incubation for 3 h. Nontreated cells were used as controls. The cell monolayer was washed three times with cold PBS and treated with trypsin/EDTA to detach the cells. The cells were collected, centrifuged, and resuspended in cold Hank’s balanced salt solution (HBSS). The cell suspensions were used for flow cytometry analysis on a BD FACSCanto II Analyzer (BD Biosciences, San Jose, CA, USA). Data were collected from at least 20 000 events. Light Sheet Microscopy. The multicellular tumor spheroids (MCTs) were obtained by using liquid overlay method. MCF-7 cells were suspended in DMEM. 200 μL of the suspension with 2 × 103 cells was seeded into each well of a U-bottom ultralow attachment 96-well plate (Corning). The plate was then centrifuged for 5 min at 500g and incubated at 37 °C with 5% CO2 for 4 days. After the incubation, cells were then incubated with micelles (1 mg mL−1) at 37 °C for 2 h. Images of MCFs were taken using a light sheet microscopy (Zeiss light sheet Z.1).

discarded and replaced with 100 μL 2× concentrated DMEM serum medium. One hundred microliters (100 μL) of micelle solution was added into each well followed by an incubation of 48 h. The plates were then washed with PBS, followed by treating with 100 μL of fresh warm medium, along with 5 μL of WST-1. The plates were then incubated for 2 h. The absorbance of the sample against the background control on a Benchmark Microplate Reader (Bio-Rad) then was read at a wavelength of 450 nm with a reference wavelength of 655 nm. The means and standard deviations were calculated by four wells under each condition in the measurement. Confocal Laser Scanning Microscopy (CLSM). MCF-7 cells were seeded in 35 mm fluorodishes at a density of 2 × 105 per dish with 2 mL culture medium and incubated for 1 day. Cells were then incubated with micelles (500 μg mL−1) at 37 °C for 3 h. After incubation, the cells were washed three times with PBS. The dishes then were mounted in PBS and observed under a laser scanning confocal microscope system (Zeiss LSM 780) equipped with a Diode 405−30 laser, an argon laser, and a DPSS 561-10 laser connected to a Zeiss Axio Observer Z.1 inverted microscope. The cell internalization was observed with a 100× 1.4 NA oil objective. The ZEN2012 imaging software (Zeiss) was used for image acquisition and processing. Flow Cytometry. Cellular association of micelles was measured using a flow cytometer (BD FACSort). Cells (MCF-7, MDA-MB-231, RAW264.7) were seeded in six-well tissue culture polystyrene plates at a density of 5 × 105 cells per well with 3 mL DMEM cell culture medium at 37 °C with 5% CO2 for 1 day prior to micelle treatment. Micelles were loaded to the cells at a working concentration of 1 mg mL−1 and then incubated for 2 h. Nontreated cells were used as controls. The cell monolayer was washed three times with cold PBS and treated with trypsin/EDTA to detach the cells. The cells were collected, centrifuged, and resuspended in cold Hank’s balanced salt solution (HBSS). The cell suspensions were used for flow cytometry analysis on a BD FACSCanto II Analyzer (BD Biosciences, San Jose, CA, USA). Data were collected from at least 20 000 events. Competition Assay with Free Fructose. MCF-7 cells were seeded in six-well tissue culture plates at a density of 5 × 105 cell per well and incubated for 2 days at 37 °C with 5% CO2 environment before fructose competition assay. Before micelle loading, the cells were treated with 0.5 mM fructose for 1 h in serum-free DMEM (with 0.1% bovine serum albumin (BSA)) and then washed with PBS twice. After that 1 mL of micelles (100 μg mL−1) were loaded along with 1 mL of serum free DMEM (with 0.5 mM fructose and 0.1% BSA) and incubated for 2 h. As the control experiment, before micelle loading, the cells were treated with serum-free DMEM (with 0.1% BSA) and then washed with PBS twice. Followed by adding 1 mL of micelles (100 μg mL−1) and 1 mL of serum free medium (with 0.1% BSA) and incubated for 2 h. The cells were then washed with cold PBS three times and collected with trypsin treatment. After trypsin removal by centrifugation, the cells were then resuspended in 1 mL of cold HBSS buffer. Flow cytometry analysis was performed using the method described as in the above sessions. Inhibition Assay (by Flow Cytometry). MCF-7 cells were seeded in six-well tissue culture plates at a density of 2 × 105 cell per well and incubated for 1 day at 37 °C with 5% CO2 environment prior to micelle treatment. Micelles were loaded to the cells at a working concentration of 250 μg mL−1 and 2× concentrated DMEM medium

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RESULTS AND DISCUSSION Polymer Synthesis. Six polymers were prepared via chain extension of PLA macro RAFT agent (Scheme 1). Two HEA polymers were synthesized, and the number of repeating units was calculated according to 1H NMR, resulting in a short chain length (PLA247-b-PHEA53) and a long chain length (PLA247-bPHEA166). The fructose polymers with two different chain lengths were prepared in a similar way. According to 1H NMR, two polymers with repeating units of 41 and 179, respectively, were obtained (PLA242-b-P[1-O-AFru]41 and PLA242-b-P[1-OAFru]179). Besides, PLA macro RAFT agent was chain extended with HEA and fructose monomers in order to generate a diblock terpolymer. The obtained polymer contained 46 units of fructose and 214 units of HEA (PLA242-b-P[1-O-AFru46-c-HEA214]). Since the polymerization rate of the fructose monomer is slightly faster than HEA, the resulting polymer will have a gradient structure with fructose being enriched toward the phenyl end functionality, resulting in micelles with higher density of fructose away from the core. In order to have all the fructose moieties located toward the shell of the micelle, a ABC triblock terpolymer was prepared. Fructose monomer was first polymerized with PLA macro RAFT agent and subsequently chain extended with HEA. According to 1H NMR, the obtained polymer had the composition PLA242-b-PHEA280-b-P[1-O-AFru]41. The synthesized polymers were characterized by NMR and SEC analysis, which is summarized in Table 1, and Figures S1 and S2 in the Supporting Information. Therefore, the PLA-b-PHEA polymers had repeating units similar to the two PLA-b-P[1-OAFru] polymers and can therefore serve as nonspecific control. The statistical polymer and triblock polymer help to answer the question if it is necessary to have a high density of fructose in the shell. D


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Figure 1. TEM images (negative staining) of the six micelles prepared: (A) MHEA53, (B) MHEA166, (C) MFru41, (D) MFru179, (E) M(Fru46co-HEA214), and (F) M(Fru41-b-HEA280). Scale bar = 0.2 μm.

Table 2. Summary of Micelle Properties parameter hydrodynamic diameter, Dh (nm) (PDI) zeta potential core radius (nm) core SLDa (A−2) shell thickness (nm) shell SLDa (A−2) solvent SLDa (A−2) aggregation number (ng) fructose numbers per micelle

MHEA53 86.5 ± 2.3 (0.08) −23.2 19.9 ± 0.8 1.36 × 10−5 5.6 ± 0.5 9.35 × 10−6 9.47 × 10−6 1347 0

MHEA166 122.4 ± 11.7 (0.12) −15.4 18.8 ± 0.4 1.36 × 10−5 10.8 ± 0.8 9.35 × 10−6 9.47 × 10−6 1139 0

MFru41

MFru179

95.6 ± 2.6 (0.13) −20.0 20.0 ± 0.5 1.36 × 10−5 6.0 ± 0.5 1.03 × 10−5 9.47 × 10−6 1400 57400

76.5 ±2.5 (0.14) −14.6 16.2 ± 0.6 1.36 × 10−5 9.4 ± 0.5 1.07 × 10−5 9.47 × 10−6 744 133176

M(Fru46-coHEA214)

M(Fru41-bHEA280)

108.7 ± 0.3 (0.15)

75.6 ± 0.6 (0.14)

−18.1 17.8 ± 0.5 1.36 × 10−5 9.7 ± 0.5 1.09 × 10−5 9.47 × 10−6 987 45402

−14.1 15.6 ± 0.5 1.36 × 10−5 11.3 ± 0.5 1.01 × 10−5 9.47 × 10−6 664 27224

a

SLD = scattering length density.

Self-Assembly into Polymeric Micelles. Polymers were initially dissolved in DMSO, followed by the dropwise addition into Milli-Q water. The obtained particles were then purified by dialysis against Milli-Q water to remove the organic solvent. The size of the micelles was determined by DLS, SAXS and TEM analysis, which were summarized in (Figure 1 and Table 2). All the micelles obtained from different polymers are named according to the composition of underpinning polymer. To be specific, HEA micelles are named as MHEA53 and MHEA166; fructose micelles are MFru41 and MFru179 and the two types of three-component micelles are M(Fru46-coHEA214) and M(Fru41-b-HEA280). According to the DLS results, a larger micelle size was obtained when increasing the chain length of the HEA block. This is in agreement with theoretical models that predict that larger micelles are generated with increasing block lengths.45 In contrast, a decreased hydrodynamic diameter with increased chain length of the fructose-based block was measured, which may be the results of strong interaction between the glycopolymers. The three-component micelles, M(Fru46-coHEA214) and M(Fru41-b-HEA280), are based on terpolymers of similar repeating units of fructose, but with different fructose distribution. Micelle M(Fru46-co-HEA214) carries the fructose pendant groups in a random manner along the hydrophilic block, resulting in micelle shells with a spread of fructose within the shell. In contrast, in the M(Fru41-b-HEA280) micelles, fructose is arranged in a block structure suggesting an enrichment of fructose on the surface of the micelle. However,

it is feasible that, due to the high compatibility of the PFru with PHEA, the two polymers do not phase-separate. Instead, the fructose-based block is most likely mixed with PHEA. The different arrangements of both polymersblock versus statisticalhave different space requirements, resulting in micelles of different hydrodynamic diameters. The sizes of these two micelles, as determined by DLS were 108 and 76 nm, respectively. The morphology of these micelles was determined by TEM and as shown in Figure 1. The nanoparticles were all found to be spherical in shape, as expected for micelles. SAXS Analysis of Micelles. While TEM and DLS can provide some information on the morphology of nanoparticles, it cannot replace in-depth characterization provided by SAXS analysis (Figure S3 in the Supporting Information and Table 2). The scattering curves in Figure S3 were fitted to a block copolymer micelle model, which is described elsewhere.46 Most importantly, the micelle resulting from the triblock terpolymer cannot be modeled with a core-two layer model, indicative of good mixing of the PHEA and the PFru block. From the fitting parameter, the aggregation number of each micelle, the size of the core, and the thickness of the corona can be calculated. In agreement with the literature, longer hydrophilic chains lead to smaller core radius, but thicker shell.45,47,48 To avoid entropically unfavorable chain stretching, longer hydrophilic chains require micelles with higher surface curvature between the hydrophobic core and the hydrophilic chain, which can be achieved by decreasing the aggregation E


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Figure 2. Illustration of the morphology of the six micelles derived from SAXS analysis.

amount of cellular association of the micelles with three different cell lines: MCF-7, MDA-MB-231, and RAW264.7 were determined using flow cytometry (Figure 3). The

number. Indeed, lower aggregation numbers were measured when the hydrophilic chain was longer. The proposed structure of each micelle is illustrated in Figure 2. Specifically, when the hydrophilic block is short (MHEA53 and MFru41), the polymers are more likely presented in a “brush” state on the surface of micelles (crew-cut micelle49) as the higher aggregation number and the lower surface curvature results in stretching of the hydrophilic chain. As a result, the tightly packed chains in MFru41 do not allow sufficient mobility and may present a hard surface in the interaction with proteins. It is known from brushes on the surface that this may lower protein binding.17,19 However, increasing the hydrophilic block chain length (MHEA166, MFru179, M(Fru46-co-HEA214) and M(Fru41-b-HEA280)), results in smaller aggregation number of the more starlike micelle and, consequently, the hydrophilic block is more likely to be in the “mushroom” state.13 The lower chain density enhances mobility, enabling the free movement of fructose within the shell, which means that, in the case of the terpolymers, the fructose is either located on the surface of the micelles or entangled toward the inner shell. Note that all polymers carry a phenyl end-functionality that is likely to interfere with the self-assembly process. However, this hydrophobic end group is present in all polymers and any effects are likely similar across all micelles. Next, the relationship between shell thickness, brush density, and fructose concentration in the shell and cellular association will be studied. In Vitro Cellular Association and Uptake. The cytotoxicity of the micelles was measured by WST-1 assay, indicating that the micelles are nontoxic to the cells at a polymer concentration up to 1 mg mL−1 (Figure S4 in the Supporting Information). The fluorescent intensity of each micelle was normalized before proceeding with the uptake test (Tables S1 and S2 in the Supporting Information). The

Figure 3. Cellular association of various micelles with three different cell lines (MCF-7, MDA-MB-231, and RAW264.7) after 2 h of incubation, measured using flow cytometry measurements. [Legend: (**) P < 0.01, (***) P < 0.001, and (****) P < 0.0001.]

interaction of micelles with cancer cells was measured to be substantially higher than with macrophages. Moreover, fructose enhanced the cellular association to MCF-7 and MDA-MB-231 breast cancer cell lines, compared to the HEA micelles, which can be assigned to the overexpression of GLUT5 transporters on the surface of breast cancer cells. Notable are the large differences in cellular association with MFru179 displaying the highest cellular uptake followed by MFru41 and then M(Fru46-co-HEA214) and M(Fru41-bF


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Figure 4. Cell uptake of micelles by MCF-7 cells after 3 h of incubation, monitored using confocal laser scanning microscopy (CLSM). Micelles were labeled with green fluorescein. Scale bar = 20 μm.

interwoven with each other. This means that similar to M(Fru46-co-HEA214), the fructose density on the surface is reduced by the presence of PHEA resulting in similar cellular uptake of M(Fru46-co-HEA214) and M(Fru41-b-HEA280). Interesting is also the comparison of MFru41 and MFru179, since both have high epitope density and should have similar uptake. However, the shorter glycoplymers led to crew-cut micelles of higher aggregation number, according to SAXS analysis. The resulting brushlike arrangement of the glycopolymer in MFru41 seems to have reduced binding to the surface receptors, which is in agreement with a detailed study on the relationship between grafting densities and lectin binding. A dense polymer layer is rather a barrier for lectin binding, because of steric hindrance.17 Moreover, it is likely that the crew-cut micelle is more dehydrated, because of the denser arrangement of polymer chains, which can be detrimental for cellular uptake as the polymer chains are less mobile.53,54 While the accessibility of the sugar by surface receptors, which is a matter of brush density and hydration, is expected to play a crucial role, the simple amount of fructose per micelle plays an important role. Three polymers have similar amounts of fructose in each polymer chain. However, the cellular uptake is significantly different. SAXS analysis revealed not only different hydration levels and brush densities, but also the aggregation number and from there the number of fructose molecules per micelle (Table 2). Correlation between cellular uptake and fructose amount per micelle revealed an almost linear dependency (Figure 5). Therefore, it seems that these parameterssugar density, hydration, polymer density cannot be discussed as independent entities and all play a role in enhancing the specific uptake by cancer cells. This complicated relationship is evident when comparing the two terpolymers, which have similar uptake, but different amounts of fructose. Sugar Competition Assay and GLUT5 Inhibition. A sugar competition assay, which uses MFru micelles and free

HEA280), which both appear to translocate into the cells at similar amounts. These results were complemented by cell uptake studies using confocal laser scanning microscopy (CLSM). Here, it can clearly be seen that the nanoparticles are internalized by the cells with MFru179 showing the highest cell internalization (Figure 4 and Figure S5 in the Supporting Information). Generally, fructose enhances cellular association, which is not surprising considering the overexpression of GLUT5 receptors in both cancerous cell lines. Cancer cells have a high affinity to glucose to compensate for the compromised oxidative phosphorylation (Warburg effect).50 Fructose has a unique role as it can ensure a high proliferation of cancer cells, even in the absence of glucose, thanks to the overexpression of GLUT5 on MCF-7 and MDA-MB-231.51 This can lead to an enhanced tumor progression in GLUT5 overexpressing tumors when fructose is present.52 It is evident that the uptake of M(Fru46-co-HEA214) and M(Fru41-b-HEA280) micelles by cancer cells is similar, albeit significantly higher than that of the MHEA micelles due to the incorporation of the fructose moiety. However, when compared to MFru41 micelles, which has the same amount of fructose as the terpolymers M(Fru46co-HEA214) and M(Fru41-b-HEA280), the terpolymers display significantly lower uptake. This means that the increased thickness of the hydrophilic shell and the dilution of fructose in a PHEA matrix did not enhance the cellular uptake. The overall low fructose density in M(Fru46-co-HEA214) micelles reduces the multivalent effect, similar to the reduced lectin binding of brushes and soluble polymers, when the epitope density is reduced.17 Surprisingly, also M(Fru41-b-HEA280) micelles, which should have a high fructose concentration in the outer layer, do not stimulate the interaction with cells. This can be explained using SAXS analysis. Although it is tempting to draw an onion-like micelle model based on ABC triblock terpolymers, SAXS analysis suggests that this micelle has a core−shell structure with the PFru and PHEA being G


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cellular uptake was less pronounced with the short fructose chain length micelles MFru41. The GLUT5 transporters are occupied with the transport of free fructose, reducing the recognition of fructose-coated micelles. While competition assays are common to prove other receptor-mediated uptake such as folate,55 the addition of fructose has many effects such as the enhanced proliferation of cell, which can hamper the conclusion. However, it is noticeable that MFru179 displays a more visible effect of cellular uptake reduction, which coincides with an overall higher cellular uptake. While this competition assay can provide a first indication that GLUT5 receptors are indeed involved in the process, this technique is prone to misinterpretation. Inhibition with (−)-epicatechin-gallate that specifically blocks GLUT5 receptors can provide a better picture.56 Using flow cytometry (Figure 6b), it becomes evident that the inhibitor reduces cellular uptake of the micelles measured. Statistical analysis can however reveal that the cellular uptake reduction is higher in fructose-coated micelles. Blocking the GLUT5 receptors will not only inhibit specific cellular uptake, but it can affect cell trafficking, cell proliferation, and the rate of apoptosis.57 Micelle Penetration into MCTs. The diffusion into 3D spheroids is governed by the ability to diffuse in the interstitial space, but also by the rate of transcellular transport. While the diffusion between cells is more prominent with very small nanoparticles, the transcellular route becomes the domineering

Figure 5. Cellular uptake of micelle by MCF-7 cells after 2 h of incubation correlated against the number of fructose per micelles, as obtained using the SAXS analysis listed in Table 2.

fructose, was employed to prove the involvement of GLUT5 transporters in the uptake process. As demonstrated in Figure 6a, free fructose in the media decreased the cellular uptake of the long fructose chain length micelles, while the reduction in

Figure 6. (a) Reduction of micelle uptake (50 μg mL−1) by MCF-7 in the presence (0.25 M) and absence of free fructose. Sugar competition assay was conducted with two MFru micelles by flow cytometry. [Legend: (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.] (b) Inhibition of micelle uptake by cytometry (−)-epicatechin-gallate (red indicates no inhibitor; blue indicates inhibitor), as measured by flow cytometry, normalized against the cellular uptake without inhibitor (set to 100%). H


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Figure 7. Light sheet images of MCF-7 spheroids treated with micelles for 2 h: (A) MHEA53, (B) MHEA166, (C) MFru41, (D) MFru179, (E) M(Fru46-co-HEA214), and (F) M(Fru41-b-HEA280). Micelles were labeled with green fluorescein. Scale bar = 100 μm.

Figure 8. Normalized fluorescence intensity distribution of micelles inside the MCF-7 spheroids: (A) MHEA53, (B) MHEA166, (C) MFru41, (D) MFru179, (E) M(Fru46-co-HEA214), and (F) M(Fru41-b-HEA280).

cellular uptake were observed. Little micelle penetration was observed for HEA micelles, followed by M(Fru46-co-HEA214) and M(Fru41-b-HEA280) micelles. The long fructose chain length micelles MFru179 showed the highest penetration into the center of the spheroid. Light sheet analysis allows the direct analysis of the layer of cells cross-sectioning the center of the spheroid (Figure S6 in the Supporting Information), showing even distribution of the micelles throughout the plane with MFru179 displaying the highest micelle concentrations. Magnification of these layers

form of penetration. Therefore, a better cellular uptake of micelles often translates into faster penetration. To test this, the micelles were incubated with MCF-7 spheroids for 2 h and analyzed using light sheet microscopy (Figure 7). In contrast to confocal fluorescent microscopy, which can only scan the surface layer of a spheroid, light sheet microscopy allows quantitative analysis capturing the whole 3D spheroid including the necrotic center (Figure 8).58−60 The results are in agreement with the in vitro cellular uptake experiment where direct correlation between spheroid fluorescence and I


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Figure 9. Illustration of interaction between fructose brush and receptors.

idea was also inspired by lectin binding studies that show that linear chains with lower epitope density often have better activity per sugar.17 Surprisingly, no improved cellular uptake was observed when comparing to MFru41. In the case of the statistical block, the hydrophilic fructose ligands were distributed randomly along the chains, placing some of the fructose close to the inaccessible core of the micelle limiting the multivalent binding of sugar moieties to the receptors. Triblock copolymer should have fared better as fructose is theoretically located on the periphery of the micelle. However, because of the compatibility of PFru and PHEA, a trilayer micelle structure is unlikely and the mobility of the long PHEA block combined with the miscibility with the PFru block led to the shelter of the fructose moieties, as revealed using SAXS studies, limiting ligand−receptor affinity. It is evident that the presence of fructose functionalities covering the nanoparticles significantly enhanced the cellular uptake. However, the polymer architectures are crucial, because the mobility, the amount of fructose per micelle, and the density of the fructose shell of micelles is instrumental. This led to the conclusion that nanoparticles require long polymer chains with high fructose density as the hydrophilic shell to achieve a high active targeting ability to the breast cancer cells. In all of these cases, the discussion around the polymer architecture cannot be separated from the micelle structure, because different block copolymers result in different aggregation numbers. In the end, it might be just as simple as increasing the numbers of fructose per micelle increasing the cellular uptake.

shows that the micelles are located inside the cells surrounded by dark intercellular space devoid of micelles, suggesting predominantly transport by the transcellular route. Proposed Model for the Interaction between Fructose Micelles and Cell Surface Receptors. In order to understand the efficiency of the interaction between fructose or nonfructose micelles with receptors, we compared the cellular uptake of MFru and MHEA, which clearly showed the significantly enhanced cellular uptake of fructose-containing micelles. Possible models for the interaction of nanoparticles with different architectures of fructose blocks with breast cancer cells are displayed in Figure 9. The polymer with the short fructose chains PLA242-b-P[1-O-AFru]41 led to crew-cut micelles with a higher aggregation number and densely packed fructose chains, which typically coincides with enhanced chain stretching. Therefore, fructose is less accessible, because the protein cannot penetrate into the high density polymer brush similar to the observations made on glycopolymer brushes grafted on surfaces.17 As a result, a lower uptake by the breast cancer cells was observed. In contrast, micelles prepared from PLA242-b-P[1-O-AFru]179 have a lower aggregation number and a larger, more hydrated glycopolymer layer. This enhances the mobility of the glycopolymer of the starlike micelle and results in easier recognition by the receptors. Although it is feasible that only a few fructose moieties on the micelles are required to enhance the cellular uptake, preparation of hydrophilic copolymers with HEA led to significantly decreased uptake and transport. The synthesized PLA242-b-P[1-O-AFru46-c-HEA214] and PLA242-b-PHEA280-bP[1-O-AFru]41 contained similar amounts of fructose per polymer as the short fructose chain length polymer (PLA242-bP[1-O-AFru]41). We originally hypothesized that increasing the hydrophilic layer and therefore the mobility of the fructose moieties by adding HEA would enhance cellular uptake. This

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CONCLUSIONS In summary, six micelles with different compositions and architectures of the hydrophilic block were prepared and analyzed using different scattering techniques. Cellular uptake studies and observation of the spheroid penetration revealed J


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Recognition: Synthesis, Self-Assembly and Lectin Binding. Macromolecules 2014, 47, 4676−4683. (10) Yilmaz, G.; Uzunova, V.; Hartweg, M.; Beyer, V.; Napier, R.; Becer, C. R. The effect of linker length on ConA and DC-SIGN binding of S-glucosyl functionalized poly(2-oxazoline)s. Polym. Chem. 2018, 9, 611−618. (11) Kumar, J.; Bousquet, A.; Stenzel, M. H. Thiol-alkyne chemistry for the preparation of micelles with glycopolymer corona: dendritic surfaces versus linear glycopolymer in their ability to bind to lectins. Macromol. Rapid Commun. 2011, 32, 1620−6. (12) Kumar, J.; McDowall, L.; Chen, G.; Stenzel, M. H. Synthesis of thermo-responsive glycopolymersviacopper catalysed azide−alkyne ‘click’ chemistry for inhibition of ricin: the effect of spacer between polymer backbone and galactose. Polym. Chem. 2011, 2, 1879−1886. (13) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.; Chen, Z.-S. The development of anticancer ruthenium(ii) complexes: from single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017, 46, 5771−5804. (14) Rosencrantz, R. R.; Nguyen, V. H.; Park, H.; Schulte, C.; Böker, A.; Schnakenberg, U.; Elling, L. Lectin binding studies on a glycopolymer brush flow-through biosensor by localized surface plasmon resonance. Anal. Bioanal. Chem. 2016, 408, 5633−5640. (15) Park, H.; Rosencrantz, R. R.; Elling, L.; Böker, A. Glycopolymer Brushes for Specific Lectin Binding by Controlled Multivalent Presentation of N-Acetyllactosamine Glycan Oligomers. Macromol. Rapid Commun. 2015, 36, 45−54. (16) von der Ehe, C.; Weber, C.; Gottschaldt, M.; Schubert, U. S. Immobilized glycopolymers: Synthesis, methods and applications. Prog. Polym. Sci. 2016, 57, 64−102. (17) Meng, X.-L.; Fang, Y.; Wan, L.-S.; Huang, X.-J.; Xu, Z.-K. Glycopolymer Brushes for the Affinity Adsorption of RCA120: Effects of Thickness, Grafting Density, and Epitope Density. Langmuir 2012, 28, 13616−13623. (18) Yang, Q.; Hu, M. X.; Dai, Z. W.; Tian, J.; Xu, Z. K. Fabrication of glycosylated surface on polymer membrane by UV-induced graft polymerization for lectin recognition. Langmuir 2006, 22, 9345−9349. (19) Kohri, M.; Sato, M.; Abo, F.; Inada, T.; Kasuya, M.; Taniguchi, T.; Nakahira, T. Preparation and lectin binding specificity of polystyrene particles grafted with glycopolymers bearing S-linked carbohydrates. Eur. Polym. J. 2011, 47, 2351−2360. (20) Serizawa, T.; Yasunaga, S.; Akashi, M. Synthesis and Lectin Recognition of Polystyrene Core−Glycopolymer Corona Nanospheres. Biomacromolecules 2001, 2, 469−475. (21) Lazarovits, J.; Chen, Y. Y.; Sykes, E. A.; Chan, W. C. W. Nanoparticle-blood interactions: the implications on solid tumour targeting. Chem. Commun. 2015, 51, 2756−2767. (22) Firkowska-Boden, I.; Zhang, X.; Jandt, K. D. Controlling Protein Adsorption through Nanostructured Polymeric Surfaces. Adv. Healthcare Mater. 2018, 7, 1700995. (23) Hadjidemetriou, M.; Kostarelos, K. Evolution of the nanoparticle corona. Nat. Nanotechnol. 2017, 12, 288. (24) Saptarshi, S. R.; Duschl, A.; Lopata, A. L. Interaction of nanoparticles with proteins: relation to bio-reactivity of the nanoparticle. J. Nanobiotechnol. 2013, 11, 26. (25) Dag, A.; Callari, M.; Lu, H.; Stenzel, M. H. Modulating the cellular uptake of platinum drugs with glycopolymers. Polym. Chem. 2016, 7, 1031−1036. (26) von der Ehe, C.; Rinkenauer, A.; Weber, C.; Szamosvari, D.; Gottschaldt, M.; Schubert, U. S. Selective Uptake of a Fructose Glycopolymer Prepared by RAFT Polymerization into Human Breast Cancer Cells. Macromol. Biosci. 2016, 16, 508−521. (27) Zhao, J.; Babiuch, K.; Lu, H.; Dag, A.; Gottschaldt, M.; Stenzel, M. H. Fructose-coated nanoparticles: a promising drug nanocarrier for triple-negative breast cancer therapy. Chem. Commun. 2014, 50, 15928−15931. (28) Majdanski, T. C.; Pretzel, D.; Czaplewska, J. A.; Vitz, J.; Sungur, P.; Höppener, S.; Schubert, S.; Schacher, F. H.; Schubert, U. S.; Gottschaldt, M. Spherical and Worm-Like Micelles from Fructose-

the importance of the structure of the fructose containing block in the shell of the micelles. It was observed that a high density of fructose combined with a long fructose containing hydrophilic block is necessary to achieve the highest possible uptake by breast cancer cells as the mobility of the long PFru enables easier receptor access. Therefore, the architecture of the glycopolymer plays a more important role as decorating a nanoparticle randomly with a few fructose groups will not be able to maximize the outcome.

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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.biomac.8b01406. 1 H NMR and SEC curves of six polymers before deprotection. SAXS fitting curves, cytotoxicity, fluorescence intensity and cellular uptake of the six micelles (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Martina H. Stenzel: 0000-0002-6433-4419 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.L. acknowledges the Chinese Scholarship Council (CSC) for scholarship support. M.S. thanks the Australian Research Council (ARC) for support. We thank the Mark Wainwright Analytical Centre for support, in particular the Biomedical Imaging Facility and the Electron Microscope Unit.

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REFERENCES

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Sugar Concentration and Arrangement on the Surface of

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