Engineered Hydrogen-Bonded Glycopolymer Capsules and Their

Interfaces , 2017, 9 (7), pp 6444–6452. DOI: 10.1021/acsami.6b15459. Publication Date (Web): February 10, 2017. Copyright © 2017 American Chemical ...
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Engineered Hydrogen-Bonded Glycopolymer Capsules and Their Interactions with Antigen Presenting Cells Kristian Kempe,*,†,‡,§ Sue D. Xiang,∥ Paul Wilson,†,§ Md. Arifur Rahim,‡ Yi Ju,‡ Michael R. Whittaker,† David M. Haddleton,†,§ Magdalena Plebanski,*,∥ Frank Caruso,*,‡ and Thomas P. Davis*,†,§ †

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia ‡ ARC Centre of Excellence in Convergent Bio-Nano Science and Technology and the Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia § Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom ∥ Department of Immunology and Pathology, Central Clinical School, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Victoria 3052, Australia S Supporting Information *

ABSTRACT: Hollow glycopolymer microcapsules were fabricated by hydrogen-bonded layer-by-layer (LbL) assembly, and their interactions with a set of antigen presenting cells (APCs), including dendritic cells (DCs), macrophages (MACs), and myeloid derived suppressor cells (MDSCs), were investigated. The glycopolymers were obtained by cascade postpolymerization modifications of poly(oligo(2-ethyl-2-oxazoline methacrylate)-stat-glycidyl methacrylate) involving the modification of the glycidyl groups with propargylamine and the subsequent attachment of mannose azide by copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC). Multilayer assembly of the hydrogen-bonding pair (glycopolymer/poly(methacrylic acid) (PMA)) onto planar and particulate supports (SiO2 particles, d = 1.16 μm) yielded stable glycopolymer films upon cross-linking by CuAAC. The silica (SiO2) particle templates were removed yielding hollow monodisperse capsules, as demonstrated by fluorescence and scanning electron microscopy. Cellular uptake studies using flow cytometry revealed the preferential uptake of the capsules by DCs when compared to MACs or MDSCs. Mannosylated capsules showed a cytokine independent cis-upregulation of CD80 specifically on DCs and a trans-downregulation of PDL-1 on MDSCs. Thus, the glycopolymer capsules may have potential as vaccine carriers, as they are able to upregulate costimulatory molecules for immune cell stimulation on DCs and at the same time downregulate immune inhibitory receptors on suppressor APC such as MDSCs. KEYWORDS: poly(2-oxazoline), mannose, layer-by-layer, dendritic cells, MDSC, macrophages, PDL-1, CD80

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INTRODUCTION Polysaccharides are naturally occurring polymers that play important roles in our immune system, e.g., in cellular recognition processes and pathogen infections. Interactions of sugar ligands (such as those expressed on bacterial cell walls) and the corresponding sugar binding proteins, so-called lectins which are often overexpressed on the surface of antigen presenting cells (APC), trigger or regulate immune responses.1,2 In recent years this specific interaction has stimulated the interest in glycomimetic materials with improved therapeutic efficiencies.3,4 Synthetic glycopolymers have been demonstrated to be superior oligosaccharide mimics because they enable the incorporation of multiple saccharides along a polymer backbone and thus amplification of carbohydrate− lectin/cell interactions,5,6 also known as the cluster-glycoside effect.7 To date, glycopolymers have been prepared using different polymerization techniques,8−12 including, but not © XXXX American Chemical Society

limited to, reversible deactivation radical polymerizations (RDRPs), ring-opening metathesis polymerization (ROMP), and ring-opening polymerizations (ROPs). The self-assembly of glycopolymers or the surface modification of particles with sugar units give rise to even more sophisticated materials, namely, glycoparticles, which not only show carbohydratespecific interactions but additionally exploit the beneficial physiochemical properties of particulate systems.13−15 To date, a wide range of glycosylated carriers has been fabricated, such as micelles, polymersomes, and metallic, magnetic, and selfassembled nanoparticles. Preferential uptake of particles by specific types of antigen presenting cells (APCs), such as macrophages (MACs), Received: December 2, 2016 Accepted: January 25, 2017

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DOI: 10.1021/acsami.6b15459 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Mannosylated PEtOx-Based Brush Copolymer (PEtOxMAMan) Synthesis: (A) Glycidyl Ring-Opening Reaction Using Propargylamine (PEtOxMAAlk); (B) Introduction of Mannose Moieties by CuAAC (PEtOxMAMan)

decade, a range of glycopoly(2-oxazolines) has been reported using glycosylated 2-oxazoline monomers40 or postpolymerization reactions such as thiol−ene chemistry.41−43 An elegant approach for the synthesis of glycosylated brush-like POx was reported by Weber et al. using telechelic POx macromonomers with α-sugar and ω-methacrylate end groups.44 In this work, we demonstrate the fabrication of LbL assembled glycopoly(2-oxazoline)-based capsules. To the best of our knowledge, hollow multilayered capsules prepared by hydrogen-bonded LbL of glycopolymers has not yet been reported. The glycopolymer was obtained by a combination of cationic ring-opening polymerization (CROP), RDRP, and highly efficient postpolymerization modifications. Alkyne functionalities introduced during this process were exploited for the modification with mannose moieties, labeling, and multilayer cross-linking. The mannosylated films and capsules showed specific interactions with the lectin concanavalin A (ConA). All capsule types tested were clearly preferentially taken up by DCs when compared to MACs or MDSCs. Moreover, the functional characteristics of the microparticles are further investigated in their capacity to downregulate PDL-1 on multiple APC types, including professional suppressor APCs such as MDSCs. Only mannosylated capsules were capable of selectively upregulating CD80 specifically on DCs (but not MDSCs or MACs). The mechanism by which these molecules are controlled was further explored by the use of fluorescently labeled microparticles, providing evidence for the existence of two mechanisms: cytokine independent cis-activation for CD80 upregulation specifically on DCs, and broad trans-downregulation of PDL-1 across multiple cell types (balanced against particle uptake induced upregulation). Together these results point to useful novel dual properties for mannosylated particles, making them suitable vaccine carriers, by virtue of being taken up by DCs and downregulating broadly immune inhibitory receptors on suppressor APCs such as MDSCs, while selectively being able to upregulate costimulatory molecules for immune cell stimulation on DCs.

dendritic cells (DCs), or myeloid derived suppressor cells (MDSCs), can lead to different immune responses and eventually different therapeutic outcomes. Uptake by DCs is usually a key step to induce adaptive immune responses and has been a major focus of synthetic vaccine development for infectious diseases and cancer.16,17 By contrast, uptake by MACs or MDSCs can lead to either activation or conversely suppression of immune responses and is being explored to develop therapeutics against autoimmune and allergic diseases.18 The state of activation of the APCs and the type of surface molecules that they upregulate after interacting with particles will also influence their ability to activate or suppress immune responses. In this context, MACs and MDSCs that express high levels of the programmed death ligand 1 (PDL-1) have high capacity to turn off immunity by interacting with programmed death 1 (PD1) molecules present on diverse immune cells such as T cells and NK cells.19 By contrast, activated mature DCs that express high levels of molecules that allow them to effectively interact with T cells, such as CD80, are classically strong activators of immunity.20 Herein, we explore the balance of preferential uptake of glycomimetic layer-by-layer (LbL) microparticles by DCs compared with MACs and MDSCs, as well as the potential change in key regulatory molecules CD80 and PDL-1 on these different APC subsets. Poly(2-oxazolines) (POx) are an emerging polymer class,21 which have gained extensive attention in the biomaterials area22,23 since they combine excellent biocompatibility24−27 and protein repellence properties28 with the ease of synthesizing highly functional materials.29−32 Recently, we described the assembly of POx-based systems using the LbL technique for the fabrication of low-fouling, redox- and enzyme-responsive, and targeted hollow polymer capsules.33−35 The LbL approach36,37 has attracted increasing interest for the engineering of particulate delivery systems,38,39 as it allows tailoring of the physical and chemical properties of the carriers. Previously, particular focus was placed on the design of brush-like POx by RDRPs as they allow for the incorporation of further functional groups that are beneficial for covalent or reversible cross-linking and labeling or modification with targeting units of the capsules postassembly. Both copper-catalyzed alkyne−azide cycloaddition (CuAAC)33,35 and dynamic thiol−disulfide exchange reactions34 were employed for the stabilization, i.e., crosslinking, of the multilayered films and for the modification of the capsule surface with an antibody for the specific targeting of activated platelets.35 Other interesting targeting ligands that are readily available are mono- and disaccharides. Over the past

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RESULTS AND DISCUSSION Copolymer Synthesis. Mannosylated brush-like PEtOxbased copolymers (PEtOxMAMan) were prepared in a multistep procedure comprising PEtOxMA macromonomer synthesis by CROP (Supporting Information Scheme S1), brush copolymer synthesis by atom transfer radical polymerization (ATRP), and efficient ring-opening reaction of glycidyl side chains and CuAAC (Scheme 1). In the first step, the CROP of 2-ethyl-2oxazoline (EtOx) was quenched with methacrylic acid to B

DOI: 10.1021/acsami.6b15459 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. (A) 1H NMR spectra (400 MHz, CDCl3) of poly[oligo(2-ethyl-2-oxazoline methacrylate)-stat-glycidyl methacrylate] (PEtOxMAGMA) (bottom), and the modification of the glycidyl moieties with propargylamine (PEtOxMAAlk) (top). (B) SEC traces (solvent, DMAc/LiBr) of PEtOxMAGMA (black solid line), PEtOxMAAlk (red long dash line), and PEtOxMAMan (blue short dashed line).

Scheme 2. Hydrogen-Bonded LbL Assembly of PEtOxMAAlk (Non-mannosylated) or PEtOxMAMan (Mannosylated) and PMA for the Preparation of the Respective PEtOxMAx Capsules

and CuAAC with mannose azide resulted in a shift of the SEC traces to higher molecular weights (Figure 1B). Multilayer Assembly on Planar and Particle Supports. The hydrogen-bonding LbL assembly of PEtOxMAAlk/PMA and PEtOxMAMan/PMA films was monitored by quartz crystal microbalance (QCM) and flow cytometry. To this end, five PEtOxMAx/PMA bilayers were iteratively deposited using polymer solutions (1 mg mL−1) in 50 mM NaOAc, pH 4. For QCM studies, the QCM crystals were first coated with a PEI (1 mg mL−1, 0.5 M NaCl) layer. Subsequently, PMA and PEtOxMAx were adsorbed for 15 and 20 min, respectively, and the films were washed into NaOAc buffer after each adsorption. Upon the individual deposition of polymers a frequency decrease was observed suggesting the formation of hydrogenbonded PEtOxMAx/PMA multilayer films (Figure S2A). The deposition of the PEtOxMAMan/PMA bilayer system showed only a slightly higher frequency change compared to the PEtOxMAAlk/PMA bilayer system, suggesting a similar hydrogen-bonding interaction of both systems. To examine the multilayer film formation on particle supports, PEtOxMAx and AF488-cadaverine labeled PMA (PMA488) were deposited alternatively onto monodisperse SiO2 particles (1.16 μm in diameter) at pH 4. Flow cytometry was used to measure the fluorescence intensity of the particles after the deposition of each bilayer. The layer buildup for both bilayer systems was regular and linear (Figure S2B) similarly to the QCM studies. For the fabrication of hollow mannosylated and nonmannosylated polymer capsules, five PEtOxMAMan/PMA and PEtOxMAAlk/PMA bilayers, respectively, were deposited onto monodisperse SiO2 particles (1.16 μm). Subsequently, the films were stabilized by cross-linking with a bisazide PEG linker similar to a procedure reported for other CuAAC “click”

prepare the PEtOx macromonomer, namely, oligo(2-ethyl-2oxazoline methacrylate) (OEtOxMA) (Scheme S1).45 Atom transfer radical copolymerization of OEtOxMA and glycidyl methacrylate (GMA) yielded highly functional copolymers (PEtOxMAGMA), which can be efficiently modified through ring-opening reaction with nucleophiles. In this way, brush-like PEtOx with pendant alkyne moieties were prepared (PEtOxMAAlk), which were further functionalized with mannose azide in a CuAAC reaction to yield PEtOxMAMan. The degree of polymerization (DP) and functionalization (DF) was determined by 1H NMR spectroscopy. EtOx was polymerized with a monomer-to-initiator [M:I] ratio of 4:1. Termination of the living chain ends with methacrylic acid upon full conversion of the monomer yielded the OEtOx4MA macromonomer. ATRP of OEtOxMA and GMA provided functional PEtOx-containing copolymers with 17 mol % glycidyl groups, as determined by the ratio of the integrals of the GMA peaks (2.5−2.9 and 4.2 ppm) and the CH2 peak of the PEtOx side chain (2.1−2.5 ppm). The complete disappearance of the GMA peaks after treatment with propargylamine suggested their quantitative modification with alkyne groups (PEtOxMAAlk; Figure 1A, top). CuAAC of the latter with mannose azide resulted in the formation of triazole groups, which was confirmed via a 1H NMR signal at 8.1 ppm (Figure S1). The comparison of the integral ratio of the triazole and the CH2 PEtOx signal revealed an overall mannose content of 6 mol %. Thus, PEtOxMAMan still contained unreacted alkyne groups, which could be used for further postpolymerization reactions, such as cross-linking reactions and labeling. In all cases, dispersity (Đ) values 18 MΩ). Mannose azide was prepared according to a literature procedure. Nuclear magnetic resonance (1H NMR) samples were measured using deuterated solvents (CDCl3, DMSO-d6) on a Varian INOVA system (400 MHz, 25 °C). Molecular weights and distributions of the polymer samples were analyzed on a Shimadzu SEC system equipped with a SIL-20AD automatic injector, a DGU-12A degasser, a 5.0 μm bead-size guard column (50 × 7.8 mm2) followed by three KF-805L columns (300 × 8 mm2; bead size, 10 μm; pore size maximum, 5000 Å), and a RID-10A differential refractive index and a SPD-20A ultraviolet detector. A CTO-20A oven controlled the temperature of the columns (40 °C). N,N-Dimethylacetamide (DMAc; HPLC grade, with 0.03% (w/v) LiBr) was used as eluent at a flow rate of 1 mL min−1. Polystyrene was used as standard (500 to 2 × 106 g mol−1). An inverted Olympus IX71 microscope containing a DIC slider (UDICT, Olympus) and a 60× objective lens (Olympus UPLFL20/0.5 N.A.; W.D., 1.6) was used for fluorescence microscopy measurements. A FEI Quanta 200 field emission scanning electron microscope (SEM) operated at an accelerating voltage of 10 kV was used to obtain SEM images. Polymer Synthesis and Postmodifications. Oligo(2-ethyl-2oxazoline methacrylate) (OEtOxMA4) and Poly(oligo(2-ethyl-2oxazoline methacrylate)-stat-glycidyl methacrylate) (PEtOxMAGMA). ([OEtOxMA]:[GMA]:[EBIB] = 60:40:1) were prepared according to a modified reported procedure.34 Characterization of OEtOxMA4. SEC (DMAc, PS standard): Mn = 1 020 g mol−1, Đ = 1.13; PEtOxMAGMA, 47% and 53% conversion for OEtOxMA and GMA. 1H NMR (400 MHz, CDCl3): δ 4.5−4.2 (COO−CH2 GMA), 4.2−3.3 (N−CH2 OEtOxMA), 3.3−3.1 (CHO GMA), 3.1−2.9 (CH3N OEtOxMA), 2.9−2.5 (CH2O GMA), 2.5−2.1 (CH2 OEtOxMA), 2.0−1.4 (CH2 methacrylate backbone), 1.3−0.9 (CH3 OEtOxMA), 0.9− 0.6 (CH3 methacrylate backbone). SEC (DMAc, PS standard): Mn = 17 500 g mol−1; Đ = 1.37. Modification of PEtOxMAGMA with Propargylamine (PEtOxMAAlk). A solution of PEtOxMAGMA (150 mg, 0.0085 mmol), propargylamine (76 mg, 1.37 mmol), and triethylamine (138 mg, 1.37 mmol) in DMSO (2 mL, anhydrous) was stirred overnight at 50 °C. After the removal of the solvent, the polymer was re-dissolved in methanol. Dialysis (MWCO = 3.5 kDa) against methanol (1 day), Milli-Q (2 days) and subsequent freeze-drying yielded PEtOxMAAlk. 1H NMR (400 MHz, CDCl3): δ 4.3−3.2 (N−CH2 OEtOxMA, COO−CH2 GMA), 3.1−2.9 (CH3N OEtOxMA), 2.5−2.1 (CH2 OEtOxMA), 2.0− 1.4 (CH2 methacrylate backbone), 1.3−0.9 (CH3 OEtOxMA), 0.9−0.6 (CH3 methacrylate backbone). SEC (DMAc, PS standard): Mn = 30 200 g mol−1; Đ = 1.28. Modification of PEtOxMAAlk with Mannose Azide (PEtOxMAMan). PEtOxMAAlk (20 mg, 0.0011 mmol) and mannose azide (2.6 mg, 0.011 mmol) were dissolved in DMF (0.5 mL), and PMDETA (3.65 mg, 0.02 mmol) was added before the solution was deoxygenated for 30 min. Subsequently, CuBr (3 mg, 0.02 mol) was added. After 19 h stirring, unreacted compounds and CuBr/PMDETA were removed by dialysis (MWCO = 3.5 kDa) against Milli-Q (3 days) and preparative SEC using a NAP-25 column. Freeze-drying yielded PEtOxMAMan. 1H NMR (400 MHz, DMSO-d6): δ 8.25−7.9 (triazole), 6.0−4.5 (mannose), 4.25−3.0 (N−CH2 OEtOxMA, COO−CH2 GMA), 3.0− 2.8 (CH3N OEtOxMA), 2.4−2.0 (CH2 OEtOxMA), 2.0−1.25 (CH2 methacrylate backbone), 1.25−0.75 (CH3 OEtOxMA), 0.75−0.5 (CH3 methacrylate backbone). SEC (THF, PS standard): Mn = 42 900 g mol−1; Đ = 1.4. Film and Capsule Preparation. Multilayer Assembly on Planar Supports. Specifications of the QCM setup and the procedure for the multilayer buildup of five PMA/PEtOxMAx bilayers can be found elsewhere.33 PMA and PEtOxMAx solutions were prepared as follows: 1 g L−1 in 20 mM NaOAc, pH 4. Multilayer Assembly on Particle Supports. This procedure is analogue to a recently reported one with the following modifications: SiO2 (1.16 um); 20 mM NaOAc was used for the aqueous solutions; particles were incubated with PMA and PEtOxMAx for 10 and 15 min, respectively; a total of four bilayers was targeted.34 G

DOI: 10.1021/acsami.6b15459 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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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/acsami.6b15459. Macromonomer synthesis scheme, 1H NMR spectrum of glycopolymer, LbL assembly data, ConA study images, and cytokine analysis data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(K.K.) E-mail: [email protected]. *(M.P.) E-mail: [email protected]. *(F.C.) E-mail: [email protected]. *(T.P.D.) E-mail: [email protected]. ORCID

Kristian Kempe: 0000-0002-0136-9403 Paul Wilson: 0000-0002-9760-899X Yi Ju: 0000-0003-0103-1207 David M. Haddleton: 0000-0002-4965-0827 Frank Caruso: 0000-0002-0197-497X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Sylvia T. Gunawan and Ka Fung Noi for their help with the preparation of glycopolymer capsules. This work was carried out within the Australian Research Council (ARC) Centre of Excellence in Convergent Bio-Nano Science and Technology (Project No. CE140100036). P.W. thanks the Leverhulme Trust for an Early Career Fellowship (ECF/2015075). K.K. gratefully acknowledges the award of a NHMRCARC Dementia Research Development Fellowship (APP1109945). M.P. is a NHMRC Senior Research Fellow (APP1059409). F.C. (FL120100030) and T.P.D. (FL140100052) are grateful for the awards of Australian Laureate Fellowships from the ARC.

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REFERENCES

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DOI: 10.1021/acsami.6b15459 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.6b15459 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX