Antifouling Peptide Dendrimer Surface of ... - ACS Publications

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Antifouling Peptide Dendrimer Surface of Monodisperse Magnetic Poly(glycidyl methacrylate) Microspheres Helena Hlídková, Ilyia Kotelnikov, Ognen Pop-Georgievski, Vladimír Proks, and Daniel Horák* Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Heyrovský Sq. 2, 162 06 Prague 6, Czech Republic S Supporting Information *

ABSTRACT: Antifouling properties and stability in tissue fluids are crucial for the successful application of micro- and nanoparticles in biomedicine. In this study, we prepared monodisperse magnetic poly(glycidyl methacrylate) microspheres with amino groups (mgt.PGMA-NH2) by a multistep swelling polymerization of glycidyl methacrylate (GMA). This was followed by ammonolysis of oxirane groups and precipitation of iron oxides inside the particle pores to make the microspheres magnetic. To suppress nonspecific protein adsorption from biological media, the microspheres were covered by three generations of a compact amino acid dendritic network (Ser-Lys-Ser/Lys-Ser/Lys-Ser) using peptide chemistry. The resulting particles did not aggregate under physiological conditions and contained ∼1 mmol of NH2/g that was available for further modifications. Alkyne groups accessible for click chemistry were introduced to the dendrimer-coated particles by a reaction with 4-pentynoic acid. The external particle surface and internal bulk were characterized by scanning (SEM) and transmission electron microscopy (TEM), atomic absorption (AAS), FTIR, X-ray photoelectron spectroscopy (XPS), and elemental analysis. Antifouling properties of the dendrimer and linear Ser-Ala-Ser/Ala-Ser/Ala-Ser peptide-modified mgt.PGMA-NH2 microspheres were challenged with solutions of proteins, such as bovine serum albumin (BSA), γ-globulin (γ-Gl), fibrinogen (Fg), and a mixture of them. Finally, a model azide−alkyne cycloaddition reaction with 125I-radiolabeled azidopentanoyl-GGGRGDSGGGY(125I)-NH2 (125I-N3-RGDS) peptide demonstrated that the dendrimer-modified particles are suitable for potential applications, including the separation of peptides and other biomolecules, diagnostics, mimetics, vaccine synthesis, etc.

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INTRODUCTION Biostable polymers with antifouling surfaces are of significant interest for biomedical applications, e.g., biosensors, implants, intraocular lenses, and drug delivery systems.1 The requirement to reduce nonspecific protein adsorption stems from the fact that the protein adsorption occurs immediately after implantation of the biomaterial in the living tissue and determines the adverse reactions of the patient’s body, such as thrombus formation and bacterial infection.2 Therefore, it is important to coat the implant surface with a natural or synthetic material that minimizes the adverse nonspecific interactions. However, coating with fibronectin, collagen, laminin, heparin, dextran, chitosan, and other natural proteins © XXXX American Chemical Society

has drawbacks, such as gradual degradation, which preclude their long-term use.3−6 In contrast, synthetic polymers, e.g., poly(ethylene glycol) (PEG), avoid these disadvantages;7−9 however, they often interact with the immune system and have a limited long-term in vivo stability.10 Alternative water-soluble polymers, including poly(2-methyl-2-oxazoline),11 poly(N-(2hydroxypropyl)methacrylamide) (PHPMA),12 its copolymer with zwitterion carboxybetaine methacrylamide,13 and complexes of poly(L-lysine) (PLL) with PHPMA, display the same Received: November 24, 2016 Revised: February 9, 2017

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DOI: 10.1021/acs.macromol.6b02545 Macromolecules XXXX, XXX, XXX−XXX

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Figure 1. Dendrimer formation from the mgt.PGMA-NH2 particle surface via peptide synthesis.

or even better protein repellence than PEG.14 Layered architectures of polymeric dendrimers with a high number of controllable peripheral functionalities, and a tendency to adopt a globular shape, introduce similar antifouling properties.15−18 Poly(amidoamine) dendrimers, coupled with oligo(ethylene glycol), resist fibrinogen adsorption under physiological pH in spite of their polycationic nature.19 In contrast, polyester dendrimers exhibit charge neutrality, but peripheral OH groups and intramolecular H-bonds induce protein adsorption.20 This trend was reversed by PEG coupling.21 Dendritic polymers also include polypropyleneimine22 and PLL,23 which can conjugate with some ligands, such as truncated antibodies,24 carbohydrate

analogues,25 and peptides.26 Peptide dendrimers, which have the advantage of precise chemical design,27 are commonly used to construct biomaterials for use in drug and gene delivery as well as vaccine preparation.28,29 Peptide dendrimers can be prepared with a divergent method in which a poly(amino acid) (PLL) serves as a branching base and peptides are stepwise coupled by solid phase peptide synthesis (SPPS). The aim of this work was to design monodisperse magnetic poly(glycidyl methacrylate) (PGMA) microspheres, which could easily be separated by a magnet, withstand protein adsorption, and are suitable for biomimetic modifications. As a microsphere coating, a dendrimer consisting of short Ser-LysB


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shaking. The mixture was repeatedly washed with water, ethanol, and water in series. The resulting particles were denoted PGMA-NH2. Iron oxide was precipitated in macroporous PGMA-NH2 microspheres according to previously published reports.34,35 Wet particles (4.2 g) were washed with Ar-purged water, separated by centrifugation (4000 rpm) for 2 min under Ar atmosphere, acidified with 0.5 M HCl (2 mL), dispersed in water (10 mL) with sonication, and separated. After resuspension in FeCl2 aqueous solution (0.01 M) under Ar atmosphere, the dispersion was sonicated for 15 min. Centrifugation/ redispersion of the microspheres in the mother liquor and fresh FeCl2 solution was repeated three times to ensure high FeCl2 saturation in the particles. They were filtered after 10 min of centrifugation (4000 rpm) and resuspended in 1.4 M NH4OH (5 mL) under an Ar atmosphere. Then, water (5 mL) was added, and the suspension was rotated (20 rpm) for 16 h. The resulting mgt.PGMA-NH2 microspheres were separated and repeatedly washed with water (12 mL each) to reach a pH of 8. Magnetite (Fe3O4) in the particles was then slowly oxidized to a more stable maghemite (γ-Fe2O3) by shaking the particle suspension (150 rpm) in water (20 mL) for 16 h under oxygen. During the reaction, water was changed six times. The precipitation procedure was repeated twice to increase the iron oxide content in the particles. To remove impurities and aggregates, the mgt.PGMA-NH2 microspheres were classified via a stainless steel sieve (25 μm mesh) in a dust-free box. Modification of Monodisperse mgt.PGMA Microspheres with Dendrimers. Amino acids were covalently attached to the monodisperse mgt.PGMA-NH2 microspheres in a stepwise (divergent) approach via peptide chemistry.36 First, Fmoc-L-Ser(tBu)-OH was bound to the particle surface, which was followed by the attachment of Fmoc-L-Lys(Fmoc)-OH containing two amino groups to enable further peptide chain branching. Ser hydrophilized the particles and facilitated chain extension, which was followed by lysine branching. Subsequent chain prolongation was controlled by Fmocand tBu-protected amino acids. In this way, the particles were modified up to the third dendrimer generation (Figure 1). Here, the procedure for the formation of the first dendrimer generation (mgt.PGMA-D1) is described. Ser, Lys, and Ser (3 mol excess each) were gradually added to wet mgt.PGMA-NH2 microspheres (2 g). Preactivation of amino acid COOH groups with DIC/OP (1:2 mol/mol; DIC/amino acid = 1:1 mol/mol) solution in DMF at RT for 20 min was followed by 20 min coupling. The microspheres were washed three times with DMF (5 mL each) for 10 min. Fmoc was removed by 20 vol % piperidine in DMF twice (3 mL each) for 10 min, and the particles were again washed with DMF. Amino groups of mgt.PGMA-D1 microspheres (0.4 g) were acetylated by 20 vol % Ac2O and 30 vol % DIPEA solution in DMF at RT for 15 min, and the particles were washed with DMF. tBu was cleaved off with 50 vol % TFA and 5 vol % TIPS in DMF at 0 °C for 30 min. Finally, the microspheres were washed with DMF and 5 vol % DIPEA in DMF to neutralize the residual TFA, and they were transferred in DMF. Analogously, second- and thirdgeneration dendrimers (particles denoted as mgt.PGMA-D2 and mgt.PGMA-D3) were obtained using the same procedure with Lys and Ser. Amino acid chains attached to the particle surface consisted of Ser-Lys-Ser/Lys-Ser/Lys-Ser. The particles were successively transferred in water and dried before analysis. For the sake of comparison, mgt.PGMA-NH2 microsphere surface was also modified with a linear Ser-Ala-Ser (particles denoted as mgt.PGMA-L1), Ser-Ala-Ser-Ala-Ser (mgt.PGMA-L2), and Ser-AlaSer-Ala-Ser-Ala-Ser peptide (mgt.PGMA-L3) using a procedure described above via SPPS, where Lys was replaced by Ala. The mgt.PGMA-D3 particles (0.2 g) containing residual NH2 groups were magnetically separated from the suspension, and alkyne groups were introduced for additional modification reactions. COOH groups of 4-pentynoic acid (3 mol excess relative to amino groups) were preactivated with DIC/OP (1:2 mol/mol) solution in DMF at RT for 20 min, and coupling was performed for an additional 30 min. The mgt.PGMA-D3-PA microspheres were washed with DMF, transferred in water, and coupled with 125I-N3-RGDS peptide via a click reaction to yield mgt.PGMA-D3-125I-N3-RGDS.

Ser/Lys-Ser/Lys-Ser amino acid chains was used. While Ser enabled prolongation of dendrimer chains, which positively affected hydrophilization, Lys ensured branching. Antifouling properties of the dendrimer-modified PGMA microspheres were compared with those of nondendritic surface containing linear Ser-Ala-Ser/Ala-Ser/Ala-Ser peptide. Finally, alkynefunctionalized dendrimer-coated particles were prepared by a reaction of activated pentynoic acid with free terminal amino groups of the dendrimer. The ability to undergo a click reaction was then confirmed by coupling with an 125I-N3-RGDS peptide.

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EXPERIMENTAL SECTION

Materials. Glycidyl methacrylate (GMA; Fluka, Buchs, Switzerland) and ethylene dimethacrylate (EDMA; Ugilor S.A., France) were purified by vacuum distillation. 2-[(Methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA) was synthesized according to an earlier described procedure.30 Sodium dodecyl sulfate (SDS), dibutyl phthalate (DBP), Methocel 90 HG [(hydroxypropyl)methylcellulose], and benzoyl peroxide (BP) were supplied by Fluka. Cyclohexyl acetate (CyAc) was prepared from cyclohexanol and acetic anhydride. Ethyl(hydroxyimino)cyanoacetate (OxymaPure; OP), N,N′diisopropylcarbodiimide (DIC), piperidine, trifluoroacetic acid (TFA), triisopropylsilane (TIPS), N,N-diisopropylethylamine (DIPEA), acetic anhydride (Ac2O), bovine serum albumin fraction V (BSA; Mw = 66 000), bovine fibrinogen (Fg; Mw = 340 000), phosphate buffered saline (PBS; pH 7.4 and 8.1), FeCl2·4H2O, solvents, and other reagents were obtained from Sigma-Aldrich (Milwaukee, WI). Dimethylformamide (DMF) was purified by vacuum distillation. Fmoc-L-Ser(tBu)-OH, Fmoc-L-Ala-OH·H2O, and Fmoc-L-Lys(Fmoc)-OH were purchased from Iris Biotech (Marktredwitz, Germany), and 4-pentynoic acid (PA) was obtained from abcr (Karlsruhe, Germany). Radiolabeling Na125I solution was from Lacomed (Ř ež, Czech Republic), and 125I-labeled azidopentanoylGGGRGDSGGGY(125I)-NH2 (125I-N3-RGDS) peptide was prepared according to published procedures.31 Bovine γ-globulin (γ-Gl; Mw = 150 000) was delivered by Serva Electrophoresis (Heidelberg, Germany) and CuSO4 by Lach-Ner (Neratovice, Czech Republic). Ultrapure Q-water, ultrafiltered on a Milli-Q Gradient A10 system (Millipore; Molsheim, France), was used in all experiments. Synthesis of Monodisperse Magnetic PGMA-NH2 Microspheres. Monodisperse macroporous PGMA-NH2 microspheres were prepared by modifying a multistep swelling polymerization method described by Ugelstad.32,33 Briefly, an emulsion of monomers (1.5 g of GMA, 0.3 g of MCMEMA, and 1.2 g of EDMA) and BP (0.03 g) in 0.1% SDS aqueous solution (7.5 mL) was prepared at 0 °C with sonication (4710 Series Cole-Parmer Ultrasonic Homogenizer; Chicago, IL; 10 W) for 3 min. A latex (1.5 mL) containing polystyrene seeds (0.3 g) was prepared by emulsifier-free emulsion polymerization,34,35 mixed with emulsion (3 mL) of DBP (1 g) in 0.25% aqueous SDS for 48 h, and added to monomer emulsion, and the mixture left to swell for 16 h with stirring (30 rpm). CyAc (4.5 g) was emulsified in 0.1% SDS aqueous solution (10 mL) at 0 °C with sonication for 3 min, and the mixture was added to the above dispersion. Swelling continued for an additional 1 h with stirring (300 rpm) in a CO2 atmosphere to prevent inhibition of subsequent polymerization with oxygen. To stabilize the dispersion, 2 wt % aqueous Methocel 90 HG solution (4 mL) was added in a CO2 atmosphere, and the mixture was agitated at RT for 1 h and polymerized at 70 °C for 16 h with stirring (500 rpm). Resulting macroporous poly(glycidyl methacrylate) (PGMA) microspheres were washed with 0.01 wt % Tween 20 and tetrahydrofuran (five times each) on a Büchner funnel and were redispersed in water. Water was removed, MCMEMA in the particles was hydrolyzed with 0.2 M NaOH aqueous solution (120 mL) at RT for 5 h with stirring (50 rpm), and the suspension was left to stand for 16 h. The microspheres were washed with water, and oxirane groups of PGMA were ammonolyzed in 26% NH4OH (50 mL) at RT for 74 h during C


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Macromolecules Characterization of Microspheres. The morphology of the microspheres was analyzed with a Quanta S200 scanning electron microscope (SEM) and Tecnai Spirit G2 transmission electron microscope (TEM; both FEI, Brno, Czech Republic). Ultrathin cross sections (60 nm) for TEM were obtained by embedding the particles in London Resin White and sectioning on a LKB III (Leica; Wetzlar, Germany) ultramicrotome. The particle size was determined from SEM micrographs using Atlas software (Tescan; Brno, Czech Republic). The number- and weight-average diameters (Dn and Dw) were calculated from at least 500 microspheres. The polydispersity index PDI = Dw/Dn, where Dw = ∑niDi4/∑niDi3 and Dn = ∑niDi/∑ni. FTIR spectra were recorded on a PerkinElmer Paragon 1000PC spectrometer with the Specac MKII Golden Gate single attenuated total reflection (ATR) system in the range of 450−4400 cm−1 with 4 cm−1 resolution and 64 scans per spectrum. The iron, carbon, and nitrogen levels in the particles were determined with a PerkinElmer 3110 atomic absorption spectrometer (AAS) and PerkinElmer 2400 CHN elemental analyzer (Waltham, MA). X-ray photoelectron (XPS) spectra were recorded on a K-Alpha+ XPS spectrometer (ThermoFisher Scientific; East Grinstead, UK) operating at 10−7 Pa. The data were acquired and processed with Thermo Avantage software. The particles were analyzed using a microfocused monochromated Al Kα X-ray radiation (400 μm spot size) with 200 eV pass energy for survey and 50 eV for high-energy resolution core level spectra. The X-ray angle of incidence was 30°, and the emission angle was normal to the surface. The K-Alpha charge dual compensation system was employed during analysis using electrons and low-energy argon ions to prevent any localized charge buildup. The high-resolution spectra were fitted and deconvoluted with Voigt profiles. Assuming a simple model of a semi-infinite solid with a homogeneous composition, the peak areas were corrected for the photoelectric cross sections,37 inelastic mean free paths of the investigated electrons,38 and transmission function of the spectrometer.39 All spectra were referenced to the C 1s peak attributed to C−C and C−H at 285.0 eV binding energy and were controlled via the wellknown photoelectron peaks of poly(ethylene terephthalate) metallic Cu, Ag, and Au. The atomic concentrations of carbon, oxygen, and nitrogen were determined from the Fe 2p, C 1s, O 1s, and N 1s photoelectron peak areas after Shirley inelastic background subtraction. To compare these concentrations with the results from elemental analysis, XPS-determined particle surface composition, expressed in atomic percent (at. %), was recalculated to the weight percent (wt %) using the relative atomic weights Ar(i) (i = Fe, C, O, N) of the individual elements. The weight of the element (i) is expressed as wt(i) = at. %(i) × Ar(i), weight of all elements wttotal = wt(i) + wt(i + 1) + wt(i + 2) + ..., and wt %(i) = wt(i)/wttotal × 100. Protein Adsorption. Protein uptake was measured to estimate the behavior of amino acid-modified microspheres in body fluids, such as blood and plasma. mgt.PGMA-NH2 (control), mgt.PGMA-D1, mgt.PGMA-D2, and mgt.PGMA-D3, as well as mgt.PGMA-L1, mgt.PGMA-L2, and mgt.PGMA-L3 particles (18 mg each), were suspended in PBS (1.8 mL; pH 7.4), magnetically separated, and redispersed in 0.2 wt % BSA solution in PBS (1.8 mL; pH 7.4), and the suspension was rotated (20 rpm) at RT for 2 h. Similarly, the particles were incubated in 0.1 wt % γ-Gl, 0.01 wt % Fg solution and a protein mixture (2 mg of BSA, 1 mg of γ-Gl, and 0.1 mg of Fg/mL). Protein concentrations in the supernatants were determined by a PerkinElmer Lambda 20 UV−vis spectrometer at 278 nm, and the protein sorption on the particles was calculated. Each experiment was performed in at least duplicate. Click Reaction of Radiolabeled N3-RGDS Peptide with mgt.PGMA-D3-PA Microspheres. A click reaction of 125I-N3RGDS peptide with mgt.PGMA-D3-PA microspheres was performed in the presence and absence of Cu2+ catalyst (control).31,40 Briefly, the particles (2 mg) were mixed with 1.4 × 10−5 mol of 125I-N3-RGDS peptide aqueous solution (400 μL) and 0.1 M sodium ascorbate solution (100 μL). The suspension was purged with nitrogen for 15 min and shaken with or without 0.05 M CuSO4 solution (4 μL) for 1 h (Figure 2). After magnetic separation and multiple washes with water, the radioactivity of the mgt.PGMA-D3-125I-N3-RGDS particles was

Figure 2. (a) Modification of mgt.PGMA-D3 microspheres with 4pentynoic acid and (b) click reaction of mgt.PGMA-D3-PA particles with 125I-N3-RGDS peptide. determined in a Bqmetr4 ionization chamber (Empos; Prague, Czech Republic) and Spectro Analyzer scintillation detector (AccuSync Medical Research; Milford, CT). To evaluate the efficiency of the click reaction from increasing the N content, the above procedure was repeated with nonlabeled N3-RGDS peptide yielding mgt.PGMA-D3N3-RGDS and amount of N compared with that in mgt.PGMA-D3-PA microspheres.

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RESULTS AND DISCUSSION Dendrimer-Modified mgt.PGMA-NH2 Microspheres. Uniform PGMA microspheres, 4.5 μm in diameter, were prepared with multistep swelling polymerization of GMA, MCMEMA (providing COOH groups after hydrolysis), and an EDMA cross-linker. Here, polystyrene seeds were imbibed with liquid porogens (DBP, CyAc, and monomers) to endow the particles with macroporosity after completion of the suspension polymerization. Subsequently, oxirane groups of PGMA were ammonolyzed, and iron oxides were repeatedly precipitated inside the particle pores.34,35 The resulting mgt.PGMA-NH2 microspheres contained 22 wt % of Fe (according to AAS), exhibited outstanding magnetic35 and mechanical properties,41 did not aggregate in aqueous media, and possessed 1.35 wt % N (from elemental analysis), corresponding to ∼1 mmol of NH2/ g that was available for further reactions. To suppress nonspecific protein adsorption, mgt.PGMA-NH2 microspheres were modified with a dendritic network of amino acids (SerLys-Ser/Lys-Ser/Lys-Ser) up to the third dendrimer generation (mgt.PGMA-D3) via SPPS (Figure 1). Protected Ser and Lys molecules enabled alternating elongation and branching of dendrimeric chains, resulting in a typical globular shape. The benefits of this approach include clean coverage of the particle surface with a well-defined complex protein-resistant 3D amino acid structure. In the following reaction of mgt.PGMA-D3 microspheres with 4-pentynoic acid (PA), alkyne groups available for click chemistry were introduced. Their ability to participate in the cycloaddition reaction with azide was confirmed on a model reaction of the mgt.PGMA-D3-PA microspheres with 125I-N3-RGDS peptide (Figure 2), which supported the suitability of dendrimer-modified particles for bioapplications. Finally, the PGMA microspheres were also coated with a linear Ser-Ala-Ser/Ala-Ser/Ala-Ser peptide. Morphology, Size, and Composition of Microspheres. SEM and TEM micrographs characterized both the external microsphere surface and internal particle bulk (Figure 3). The D


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Figure 3. (a−d, f) SEM and (e) TEM (cross-section) micrographs of (a) PGMA-NH2, (b) mgt.PGMA-NH2, (c) mgt.PGMA-D1, (d, e) mgt.PGMAD2, and (f) mgt.PGMA-D3 microspheres.

Table 1. Characteristic Properties of Starting and Dendrimer-Modified mgt.PGMA Microspheres particles

Dna (μm)

PDIb

Fec (wt %)

Cd (wt %)

Od (wt %)

Nd (wt %)

CRe (wt %)

ORe (wt %)

NRe (wt %)

PGMA PGMA-NH2 mgt.PGMA-NH2 mgt.PGMA-D1f mgt.PGMA-D2g mgt.PGMA-D3h

4.5 4.5 4.7 4.8 4.8 4.8

1.01 1.01 1.03 1.08 1.01 1.03

0.0 0.0 22.4 22.6 22.5 18.4

58.6 52.6 35.9 39.4 39.6 40.8

34.3 37.9 35.6 31.5 30.8 32.7

0.0 1.9 1.4 2.0 2.4 2.9

58.6 52.6 46.3 50.9 51.1 50.0

34.3 37.9 45.9 40.7 39.7 40.1

0.0 1.9 1.7 2.5 3.1 3.6

a Number-average diameter (SEM). bPolydispersity index (SEM). cFrom AAS. dFrom elemental analysis (for the sake of comparison with XPS, the O content was obtained by subtracting C, H, N, and Fe from 100 wt %). eRecalculated without Fe. fFirst dendrimer generation. gSecond dendrimer generation. hThird dendrimer generation.

vibrations, such as amide A and B bands (N−H stretching) at 3293 and 3088 cm−1, respectively, amide II (in-plane N−H bending and C−N stretching) at 1531 cm−1, amide III at 1295 and 1232 cm−1, and amide V (out-of-plane N−H wagging) at 667 cm−1. This was accompanied by the appearance of aliphatic side chain peaks at 2972, 1426, 1370, and 1190 cm−1 (and 1085 cm−1), corresponding to C−H stretching, C−H bending, C−H methyl rocking, and C−C stretching, respectively.44,45 In the spectrum of mgt.PGMA-D3-PA microspheres (Figure 4g), the following alkyne bands were found: C−H stretch broadening of the amide A band at 3293 cm−1 and strong C−H bending at 622 cm−1 with the 1255 cm−1 first overtone.31,45 It can thus be concluded that the particle modification with the peptides was confirmed. ATR FTIR spectra of Ser, Ala, linear peptidemodified and unmodified mgt.PGMA-NH2 microspheres, and differential spectra of mgt.PGMA-L1, mgt.PGMA-L2, mgt.PGMA-L3, mgt.PGMA-D3-PA, and mgt.PGMA-D3-N3RGDS minus mgt.PGMA-NH2 are given in the Supporting Information (Figure S1). According to AAS, the iron content in the starting mgt.PGMA-NH2, mgt.PGMA-D1, and mgt.PGMA-D2 microspheres (∼22 wt %) decreased to 18 wt % in mgt.PGMA-D3

starting nonmagnetic, as well as mgt.PGMA-NH2, mgt.PGMAD1, mgt.PGMA-D2, and mgt.PGMA-D3 microspheres, had similar morphology with size increasing from 4.5 to 4.8 μm due to chemical modification (Figure 3a−d,f and Table 1). The low value of the polydispersity index (PDI < 1.05) documented uniformity of the particle size, which is very important for maintaining unique controlled and reproducible physicochemical and biochemical properties.33,42 Iron oxide was embedded inside the porous structure of the microspheres and close to their surface (Figure 3e), providing them with superparamagnetic properties.43 ATR FTIR spectra of starting mgt.PGMA-NH2 microspheres, Ser, Lys, and differential spectra of mgt.PGMA-D1, mgt.PGMA-D2, mgt.PGMA-D3, and mgt.PGMA-D3-PA minus mgt.PGMA-NH2 are shown in Figure 4. In contrast to the spectrum of starting particles, typical amidic bands in the spectra of dendrimer-modified microspheres indicated binding of Ser and Lys to the particle surface. A strong amide I band (CO stretching) at 1650 cm−1 with an intensity increasing from mgt.PGMA-D1 to mgt.PGMA-D3 indicated growing dendrimer content on the particles (Figure 4d−f). Gradually increasing peak intensity was also observed for other amide E


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Figure 4. ATR FTIR spectra of (a) mgt.PGMA-NH2 microspheres, (b) Ser, and (c) Lys. Differential spectra of (d) mgt.PGMA-D1, (e) mgt.PGMAD2, (f) mgt.PGMA-D3, and (g) mgt.PGMA-D3-PA minus mgt.PGMA-NH2.

Table 2. Content of Elements Determined by XPS particles PGMA PGMA-NH2 mgt.PGMA-NH2 mgt.PGMA-D1 mgt.PGMA-D2 mgt.PGMA-D3 mgt.PGMA-L1 mgt.PGMA-L2 mgt.PGMA-L3 mgt.PGMA-D3-PA mgt.PGMA-D3-N3-RGDS a

Fe 2p (wt %) 0 0 16.2 11.0 13.7 11.7 15.4 10.3 14.5 9.9 13.3

± ± ± ± ± ± ± ± ±

3.3 2.0 2.4 2.2 1.6 1.4 2.6 2.6 1.1

C 1s (wt %) 63.5 61.7 50.8 55.3 51.4 54.8 45.2 56.3 47.7 57.8 46.1

± ± ± ± ± ± ± ± ± ± ±

0.4 1.2 1.1 2.5 1.7 1.4 3.1 2.1 4.6 4.5 2.0

O 1s (wt %) 36.5 36.5 32.2 32.4 32.4 31.0 37.8 31.5 35.7 30.2 33.9

± ± ± ± ± ± ± ± ± ± ±

N 1s (wt %)

0.6 0.4 1.2 2.0 1.1 1.3 1.6 0.5 1.7 1.2 0.7

0 1.8 0.8 1.3 2.5 2.6 1.7 1.9 2.1 2.0 6.7

± ± ± ± ± ± ± ± ± ±

0.5 0.1 0.7 0.6 0.8 0.3 0.3 0.4 0.9 0.7

N/C 0 0.03 0.02 0.02 0.05 0.05 0.04 0.03 0.04 0.04 0.14

CR 1sa (wt %) 63.5 61.7 60.6 62.1 59.6 62.0 53.4 62.8 53.2 65.3 53.1

± ± ± ± ± ± ± ± ± ± ±

0.4 1.2 1.3 2.8 1.9 1.6 2.7 1.3 2.5 2.5 1.6

OR 1sa (wt %) 36.5 36.5 38.5 36.4 37.5 35.1 44.7 35.2 44.0 32.8 39.1

± ± ± ± ± ± ± ± ± ± ±

0.6 0.4 1.4 2.3 1.2 1.5 2.6 1.1 2.2 1.8 1.0

NR 1sa (wt %) 0 1.8 0.9 1.5 2.9 2.9 2.0 2.1 2.8 1.9 7.8

± ± ± ± ± ± ± ± ± ±

0.5 0.1 0.8 0.7 0.9 0.3 0.3 0.4 0.8 0.8

Calculated without Fe; XPS values are given as the means from eight measurements.

significant decrease in the surface iron concentration due to presence of dendrimer coating (Table 2). As the dendritic layer on the particle surface consists of carbonaceous chains (Figure 1), it is not surprising that mgt.PGMA-D1, mgt.PGMA-D2, and mgt.PGMA-D3 particles contained ca. 2 wt % more C than the starting mgt.PGMA-NH2. The XPS spectra also confirmed a gradual increase in the nitrogen content, which is expressed as the N/C ratio (Table 2); this gradual increase was in agreement with the CHN analysis. The high resolution C 1s and N 1s XPS spectra provided a more detailed view of the particle functionalization (Figure 5). The C 1s envelope of the PGMA, PGMA-NH 2 , mgt.PGMA-NH 2 , mgt.PGMA-D1, mgt.PGMA-D2, and mgt.PGMA-D3 microspheres included sp3 carbon contributions C−C, C−H (285.0 eV), C*−COO (285.6 ± 0.2 eV), C−O of hydroxyl and C(O)−O−C moieties (287.0 ± 0.3 eV), C−O−C of oxiranes (287.8 ± 0.3 eV), C(O)−O of esters (289.2 ± 0.6 eV), and C(O)−OH of carboxyls (290.6 ± 0.2 eV; Figure 5). In addition to the starting PGMA microspheres, the spectra contained the C−N contribution of amines at 286.3 ± 0.2 eV. Immobilization of the dendritic structures on the mgt.PGMA-NH2 microspheres was

particles (Table 1). If the Fe content was recalculated to account for γ-Fe2O3 and/or Fe3O4, the microspheres contained 24−32 wt % of iron oxide, which was sufficient for rapid and easy separation of the particles on a permanent magnet. To facilitate the comparison of the microspheres with different Fe, C, O, and N contents, the results from elemental analysis were recalculated assuming the absence of Fe (see CR, OR, and NR in Table 1). Dendrimer-modified magnetic particles contained by 4−5 wt % of carbon, which was more than the starting mgt.PGMA-NH2 microspheres. At the same time, the N content in mgt.PGMA-D1, mgt.PGMA-D2, and mgt.PGMAD3 microspheres increased from 2.5 to 3.6 wt %, while the starting mgt.PGMA-NH2 only had 1.7 wt % of N. This is additional confirmation of dendrimer formation on the mgt.PGMA-NH2 surface. The mgt.PGMA-D3-PA particles then contained 17.3 wt % Fe and 2.86 wt % N (NR = 3.46 wt %), which is in good agreement with the above results. In contrast to elemental analysis, XPS had a more than 10 wt % higher carbon concentration (Table 2), which was mainly because the technique only measures the top 7−12 nm of the material.46 The increased carbon content was accompanied by a F


Article

Macromolecules

D3-PA microspheres (1.9 wt %) corroborated then efficient course of the click reaction. Adsorption of Proteins on Dendrimer-Modified Magnetic PGMA Microspheres. Sorption of BSA, γ-Gl, Fg, and their mixture on mgt.PGMA-NH2, mgt.PGMA-D1, mgt.PGMA-D2, and mgt.PGMA-D3 microspheres served as a model situation in a living organism in which any material is subjected to strong nonspecific protein adsorption. Our results were compared with those obtained for PEG48 and linear peptide-coated magnetic PGMA microspheres (mgt.PGMAPEG, mgt.PGMA-L1, mgt.PGMA-L2, and mgt.PGMA-L3) (Table 3). It should be noted that the dendrimer-coated Table 3. Sorption of Proteins on mgt.PGMA-NH2, Dendrimer, and PEG-Modified mgt.PGMA Microspheres particles

BSA (mg/g)

γ-Gl (mg/g)

Fg (mg/g)

protein mixturea (mg/g)

mgt.PGMA-NH2 mgt.PGMA-D1 mgt.PGMA-D2 mgt.PGMA-D3 mgt.PGMA-PEGb mgt.PGMA-L1 mgt.PGMA-L2 mgt.PGMA-L3

32.2 13.3 15.6 18.6 15.5 17.0 18.4 30.2

25.5 8.0 7.8 9.4 14.0 7.3 7.7 9.0

9.1 6.2 5.9 5.2 8.8 5.8 5.8 6.4

64.2 27.2 28.0 22.8 27.0 30.5 42.5 39.8

a Mixture of BSA, γ-Gl, and Fg (2 + 1 + 0.1 mg/mL). bFrom ref 48. BSA = bovine serum albumin, γ-Gl = γ-globulin, and Fg = fibrinogen.

Figure 5. High resolution C 1s (left) and N 1s (right) XPS spectra of (a) PGMA, (b) PGMA-NH2, (c) mgt.PGMA-NH2, (d) mgt.PGMAD1, (e) mgt.PGMA-D2, and (f) mgt.PGMA-D3 microspheres. Black: measured data; red: fitted data; blue: contribution of functional groups on the particle surface.

particles adsorbed 2−3 times fewer proteins than the mgt.PGMA-NH2 microspheres. The sorption of BSA and γGl on mgt.PGMA-D1, mgt.PGMA-D2, and mgt.PGMA-D3 particles increased successively due to the gradually increasing thickness of the dendritic layer. The opposite tendency was observed for adsorption of Fg and the protein mixture, which was probably due to steric hindrances of large Fg molecules. The sorption of γ-Gl and Fg on dendrimer-coated magnetic particles was by 30−40% lower than that on mgt.PGMA-PEG and comparable with linear peptide-modified mgt.PGMA. The BSA sorption on mgt.PGMA-D1 and adsorption of protein mixture on mgt.PGMA-D3 particles were 14 and 16% lower compared to those on mgt.PGMA-PEG. As expected, the particles with dendritic surface repelled both BSA and protein mixture by 15−38 and 11−43% more, respectively, than the mgt.PGMA-L microspheres (Figure 6). This indicated that the dendritic layer on magnetic polymer particles significantly improved their antifouling properties in complex biological media. Click Reaction of 125I-N3-RGDS with mgt.PGMA-D3-PA Microspheres. Highly reactive alkyne groups were introduced on mgt.PGMA-D3 particles via coupling with DIC/OPpreactivated PA. The ability of mgt.PGMA-D3-PA microspheres to undergo a click reaction, which is widely utilized for highly selective separations of biomolecules, was documented on a model reaction with easily detectable 125I-N3-RGDS peptide. The initial peptide amount (0.7 × 10−5 mol/mg of particles) in the reaction stoichiometrically corresponded to the number of alkyne groups (Figure 1). Radioactivity measurements indicated that 3.8 × 10−7 mol of the 125I-N3-RGDS peptide was covalently bound per mg of particles by Cu2+catalyzed azide−alkyne cycloaddition and 0.8 × 10−7 mol was nonspecifically adsorbed in the absence of CuSO4 catalyst (Table 4). This means that 0.4 × 10−3 mol of −CCH groups

confirmed by the appearance of amide CO−NH contributions (288.7 ± 0.1 eV) in the high resolution C 1s spectra of mgt.PGMA-D1, mgt.PGMA-D2, and mgt.PGMA-D3 particles (Figure 5d−f). The weak contributions at 284.4 ± 0.1 and 292.2 ± 0.2 eV in the C 1s spectra of the dendrimer-modified particles were assigned to the presence of fluorenyl groups from incomplete Fmoc deprotection and possible TFA residues after tBu cleavage, respectively. This is a consequence of the divergent dendrimer synthesis, as reported elsewhere.47 Successful modification of the mgt.PGMA-NH2 particles with dendrimers was further corroborated by the high resolution N 1s spectra (Figure 5). While the spectrum of starting PGMA microspheres lacked any nitrogen signals, the spectra of PGMA-NH2 and mgt.PGMA-NH2 particles showed contributions of R−NH2 from amines (399.6 ± 0.1 eV) and R−NH3+ from protonated amines (402.0 ± 0.1 eV). Immobilization of the dendritic peptide structures on the magnetic particle surface led to the appearance of contributions at 400.4 ± 0.1 eV that are characteristic of the C(O)−NH amide group. In accordance with the C 1s spectra, the contribution at 403.0 ± 0.1 eV was ascribed to quaternary nitrogen formed after tBu cleavage. Notably, the amide group contributions gradually increased with every new dendritic generation that formed. XPS spectra of mgt.PGMA-L1, mgt.PGMA-L2, and mgt.PGMA-L3 microspheres (see Supporting Information) documented gradual increase of C and N contents and N/C ratio, which confirmed successful immobilization of peptide chains on the particles (Table 2 and Figure S2). Decrease of N in mgt.PGMA-D3-PA compared to mgt.PGMA-D3 confirmed successful introduction of alkyne groups. Higher amount of N in mgt.PGMA-D3-N3-RGDS (7.8 wt %) than in mgt.PGMAG


Article

Macromolecules

which is however unrealistic, because XPS measures only the surface with 10−30% error. We could not also differentiate between specific and nonspecific binding of RGDS due to low intensity of the XPS signals coming from triazole ring and nonreacted azide groups. Assuming ∼10% nonspecific peptide sorption (Table 4), reaction efficiency could be estimated to 60%, which is consistent with radioactivity measurements of mgt.PGMA-D3-125I-N3-RGDS microspheres. It can thus be concluded that the azide−alkyne cycloaddition reaction on the mgt.PGMA-D3-PA particles was successful.

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CONCLUSIONS In this report, we present a preparation of monodisperse magnetic poly(glycidyl methacrylate) (PGMA) microspheres with iron oxides embedded within a polymer bulk and surface modification of the particles by an antifouling dendrimer layer. Glycidyl methacrylate was used as a monomer due to the presence of reactive oxirane groups, enabling additional functionalization with amino, carboxyl, hydroxyl, halide, cyano, mercapto, nitro, and other groups.49 To obtain mechanically robust and porous particles, ethylene dimethacrylate cross-linking agent and inert low-molecular-weight liquids were selected as a comonomer and porogens, respectively. The spherical shape, micrometer size, and particle porosity are advantageous in terms of the high surface area available for prospective modifications. Monodisperse PGMA microspheres were produced by sophisticated multiple swelling polymerization accompanied by cleaning steps between the reaction stages. The size uniformity renders the particles with identical physical, chemical, and biological properties without any aggregation in liquids. These properties are impossible to achieve for particles that have a broad size distribution when prepared by a conventional suspension polymerization. Magnetic PGMA microspheres were then obtained by iron oxide precipitation within the particle pores. The demand for such particles is still growing because they allow for rapid and easy magnetic isolation of cells and various biological compounds from complex samples. To create a reactive surface on the particles, which would be sufficiently large for immobilization of biologically active compounds and would simultaneously suppress nonspecific protein sorption, the mgt.PGMA microspheres were modified with three generations of poly(amino acid) dendrimers. Dendrimer-modified magnetic PGMA microspheres were 16 and 43% more resistant to protein adsorption than the PEGylated and linear peptidecovered microbeads, respectively.48 Compared to the nondendritic structure, we demonstrated the advantage of the peptide dendrimers on the surface of the microspheres in terms of improved antifouling properties. Moreover, the combination of highly branched dendrimers and peptides provided multiple binding sites, which could increase the reactivity and enable possible parallel reactions with many cellular receptors. This surface may also mimic compounds in living organisms and increase the microsphere biocompatibility. Finally, 4-pentynoic acid-modified dendrimer-coated mgt.PGMA microspheres were reacted with radiolabeled azido-RGDS peptide. This simple, one-pot click reaction exhibits many benefits, such as the lack of a need for extensive purifications, presence of harmless byproducts, possibility to work in aqueous media under physiological conditions, high chemical yield, reaction specificity, and irreversibility. Alkynecontaining dendrimeric mgt.PGMA microspheres may potentially be used for highly selective click capture of targets in

Figure 6. Adsorption of BSA, γ-Gl, Fg, and their mixture on various microsphere types.

was available per gram of mgt.PGMA-D3-PA microspheres for further modification with biologically active compounds. Table 4. Level of 125I-N3-RGDS Peptide Covalently and Nonspecifically Bound to mgt.PGMA-D3-PA Microspheres via Cu2+-Catalyzed and Noncatalyzed Click Reactions, Respectively immobilization

RGDSa (mol/mg)

RGDSa (mol %)

recalculated RGDSb (mol %)

covalent nonspecific

(3.85 ± 1.47) × 10−7 (0.77 ± 0.06) × 10−7

5.5 ± 2.10 1.1 ± 0.09

47 9

0.7 × 10−5 mol 125I-N3-RGDS/mg of particles in the initial solution. According to the N content in dendrimer; measurements were performed in triplicate.

a b

To assess the efficiency of the click reaction, the number of alkyne groups on the mgt.PGMA-D3-PA microspheres was calculated from the nitrogen content determined by CHN and XPS analyses as N R in mgt.PGMA-D3 minus N R in mgt.PGMA-NH2 (Tables 1 and 2). Dendrimer coating contained 1.9 wt % N, which was 8.6 times less than that in theoretically fully developed dendrimers (16.4 wt % N; Figure 1). Because FTIR and XPS analyses confirmed the gradual development of dendrimer layers, it can be presumed that fewer dendrimers were formed on the mgt.PGMA-NH2 particle surface than expected as a result of steric hindrances. The estimated number of available alkyne groups on the mgt.PGMA-D3-PA (8.1 × 10−7 mol/mg) was then also lower than the theoretical number (0.7 × 10−5 mol/mg of particles), and peptide was thus in excess during the reaction (Table 4). The level of covalently and nonspecifically attached 125I-N3RGDS peptide on the mgt.PGMA-D3-125I-N3-RGDS microspheres reached 47 and 9 mol %, respectively. These values, though higher than the measured ones (Table 4), are in agreement with the published results.31 The efficacy of the click reaction was also estimated from the increase of N content in XPS spectra of mgt.PGMA-D3-N 3 -RGDS relative to mgt.PGMA-D3-PA microspheres (5.9 wt %), which was almost identical to the theoretically calculated value (5.8 wt %). It can be thus supposed that ∼100% reaction efficiency was reached, H


Article

Macromolecules living cells, attachment of probes for fluorescence spectroscopy, incorporation of artificial amino acids into proteins, peptides, carbohydrates, drugs, antibodies, and nucleotides, etc.

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(11) Konradi, R.; Pidhatika, B.; Mühlebach, A.; Textor, M. Poly-2methyl-2-oxazoline: A peptide-like polymer for protein-repellent surfaces. Langmuir 2008, 24, 613−616. (12) Vorobii, M.; de los Santos Pereira, A.; Pop-Georgievski, O.; Kostina, N. Y.; Rodriguez-Emmenegger, C.; Percec, V. Synthesis of non-fouling poly[N-(2-hydroxypropyl)methacrylamide] brushes by photoinduced SET-LRP. Polym. Chem. 2015, 6, 4210−4220. (13) Riedel, T.; Surman, F.; Hageneder, S.; Pop-Georgievski, O.; Noehammer, C.; Hofner, M.; Brynda, E.; Rodriguez-Emmenegger, C.; Dostálek, J. Hepatitis B plasmonic biosensor for the analysis of clinical serum samples. Biosens. Bioelectron. 2016, 85, 272−279. (14) Oupický, D.; Howard, K. A.; Koňaḱ , Č .; Dash, P. R.; Ulbrich, K.; Seymour, L. W. Steric stabilization of poly-L-lysine/DNA complexes by the covalent attachment of semitelechelic poly[N-(2hydroxypropyl)methacrylamide]. Bioconjugate Chem. 2000, 11, 492− 501. (15) Lee, C. C.; MacKay, J. A.; Fréchet, J. M. J.; Szoka, F. C. Designing dendrimers for biological applications. Nat. Biotechnol. 2005, 23, 1517−1526. (16) Liu, M.; Fréchet, J. M. J. Designing dendrimers for drug delivery. Pharm. Sci. Technol. Today 1999, 2, 393−401. (17) Yeh, P.-Y. J.; Kainthan, R. K.; Zou, Y.; Chiao, M.; Kizhakkedathu, J. N. Self-assembled monothiol-terminated hyperbranched polyglycerols on a gold surface: A comparative study on the structure, morphology, and protein adsorption characteristics with linear poly(ethylene glycol)s. Langmuir 2008, 24, 4907−4916. (18) Kozak, D.; Surawski, P.; Thoren, K. M.; Lu, C.-Y.; Marcon, L.; Trau, M. Improving the signal-to-noise performance of molecular diagnostics with PEG-lysine copolymer dendrons. Biomacromolecules 2009, 10, 360−365. (19) Yam, C. M.; Deluge, M.; Tang, D.; Kumar, A.; Cai, C. Preparation, characterization, resistance to protein adsorption, and specific avidin-biotin binding of poly(amidoamine) dendrimers functionalized with oligo(ethylene glycol) on gold. J. Colloid Interface Sci. 2006, 296, 118−130. (20) Benhabbour, S. R.; Liu, L.; Sheardown, H.; Adronov, A. Protein resistance of surfaces prepared by chemisorption of monothiolated poly(ethylene glycol) to gold and dendronization with aliphatic polyester dendrons: Effect of hydrophilic dendrons. Macromolecules 2008, 41, 2567−2576. (21) Benhabbour, S. R.; Sheardown, H.; Adronov, A. Protein resistance of PEG-functionalized dendronized surfaces: Effect of PEG molecular weight and dendron generation. Macromolecules 2008, 41, 4817−4823. (22) Stasko, N. A.; Johnson, C. B.; Schoenfisch, M. H.; Johnson, T. A.; Holmuhamedov, E. L. Cytotoxicity of polypropylenimine dendrimer conjugates on cultured endothelial cells. Biomacromolecules 2007, 8, 3853−3859. (23) Al-Jamal, K. T.; Al-Jamal, W. T.; Akerman, S.; Podesta, J. E.; Yilmazer, A.; Turton, J. A.; Bianco, A.; Vargesson, N.; Kanthou, C.; Florence, A. T.; Tozer, G. M.; Kostarelos, K. Systemic antiangiogenic activity of cationic poly-L-lysine dendrimer delays tumor growth. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 3966−3971. (24) Bowersock, T. L.; Sobecki, B. E.; Terrill, S. J.; Martinon, N. C.; Meinert, T. R.; Leyh, R. D. Efficacy of a multivalent modified-live virus vaccine containing a Mannheimia haemolytica toxoid in calves challenge exposed with Bibersteinia trehalosi. Am. J. Vet. Res. 2014, 75, 770−776. (25) Lepenies, B.; Lee, J.; Sonkaria, S. Targeting C-type lectin receptors with multivalent carbohydrate ligands. Adv. Drug Delivery Rev. 2013, 65, 1271−1281. (26) Niidome, T.; Yamauchi, H.; Takahashi, K.; Naoyama, K.; Watanabe, K.; Mori, T.; Katayama, Y. Hydrophobic cavity formed by oligopeptide for doxorubicin delivery based on dendritic poly(Llysine). J. Biomater. Sci., Polym. Ed. 2014, 25, 1362−1373. (27) Lamanna, G.; Grillaud, M.; Macri, C.; Chaloin, O.; Muller, S.; Bianco, A. Adamantane-based dendrons for trimerization of the therapeutic P140 peptide. Biomaterials 2014, 35, 7553−7561.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.6b02545.

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Figures S1 and S2 (PDF)

AUTHOR INFORMATION

Corresponding Author

*(D.H.) E-mail [email protected]; Tel +420 296 809 260. ORCID

Vladimír Proks: 0000-0001-7368-7120 Daniel Horák: 0000-0002-6907-9701 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of the Czech Science Foundation (GAČ R; No. 16-01128J) is gratefully acknowledged. The study was also partially supported by GAČ R projects No. 1509368Y and 16-02702S and OPPK project No. CZ.2.16/ 3.1.00/21545 from the European Regional Development Fund.

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