Article Cite This: Macromolecules XXXX, XXX, XXX−XXX
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Comblike Polymers with Topologically Different Side Chains for Surface Modification: Assembly Process and Interfacial Physicochemical Properties Shivaprakash N. Ramakrishna,*,† Giulia Morgese,†,‡ Marcy Zenobi-Wong,‡ and Edmondo M. Benetti*,† Polymer Surfaces Group, Laboratory for Surface Science and Technology, Department of Materials, and ‡Tissue Engineering and Biofabrication, Department of Health Sciences and Technology, ETH Zürich, Zürich, Switzerland
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S Supporting Information *
ABSTRACT: The effects of side-chain topology within comblike polymers (CLPs) are revealed when these are assembled on inorganic surfaces to yield dense brush interfaces. CLPs featuring cyclic poly(2alkyl-2-oxazoline) (PAOXA) side chains and a surface-interacting backbone are less sterically hindered with respect to their linear graftbearing analogues, show slower adsorption kinetics, and generate denser assemblies on TiO2 surfaces. Cyclic PAOXA brush interfaces generated from the CLP assembly showed a molecularly smooth and compact nanomorphology, which provides extraordinary lubrication properties to the films when these are sheared against a bare inorganic surface as well as an identical polymer layer, and quantitatively prevent surface contamination within serum-rich media. Topology effects by cyclic segments within highly branched copolymer architectures introduce a new class of surface modifiers, which are broadly applicable in the designing and modification of a variety of materials.
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INTRODUCTION After having been considered as little more than a scientific curiosity for more than two decades, cyclic polymers have been recently emerging as starting compounds to fabricate polymeric materials with advanced and often unprecedented physicochemical properties. By virtue of the fundamental advances in their synthesis both by ring expansion and ring closure strategies,1−6 as well as the progresses in the full dissection of their solution and bulk properties,7−13 cyclic polymers have been nowadays featured within several materials formulations, including, among others, block copolymer micelles,14−17 core−shell nanoparticles (NP),18 macromolecular vehicles for drug19 and gene delivery,20,21 and antifouling coatings.22−24 We have been particularly interested in investigating how polymer topology effects typically observed in solution translate into interfacial properties by assemblies of cyclic homopolymer adsorbates grafted on flat, macroscopic surfaces and NPs. Upon comparison of chemically identical, linear analogues, the obtained cyclic polymer “brushes” displayed a more compact structure that guaranteed improved steric stabilization and biopassivity toward complex protein mixtures.23,24 In addition, the intrinsic absence of dangling linear chains hindered interpenetration between opposing cyclicbrush surfaces when sheared against each other, substantially improving their lubrication properties.24 Having established how cyclic homopolymer adsorbates generate brush films with improved interfacial physicochemical © XXXX American Chemical Society
properties with respect to their linear counterparts, we turned our attention to the designing of surface modifiers based on comblike polymers (CLPs) bearing cyclic side chains. Similar CLPs featuring hydrophilic and bioinert linear grafts and a surface-reactive backbone have become widespread additives for the modification of a large variety of biomaterials and have already entered in the market. In particular, CLPs have been applied to functionalize inorganic,25 organic,26,27 and polymeric supports28,29 by simple dip-and-rinse procedures, yielding antifouling coatings,30−33 biofunctional surfaces for cell manipulation,34,35 and lubricious interfaces.36 Moreover, their application has been further expanded to the modification of biosensors,37,38 to coat microfluidic devices 39 and colloids,40,41 and as scaffolds for gene therapy.42,43 Hence, considering the versatility and broad applicability of their linear side-chain-bearing analogues, CLPs including cyclic grafts would represent a structurally new class of surface modifiers, which could be applied within all the abovementioned formulations while possessing a similar chemical composition but different polymer topology-dependent physicochemical properties. Besides their extremely broad applicability, the synthesis of CLPs featuring cyclic side chains and the comprehensive dissection of their properties, especially when applied on Received: November 30, 2018 Revised: January 24, 2019
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DOI: 10.1021/acs.macromol.8b02549 Macromolecules XXXX, XXX, XXX−XXX
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1.18 mmol) was added to the polymerization mixture, which was kept at 70 °C under an Ar atmosphere for ∼20 h. After this time, the polymerization was terminated by adding an aqueous solution of Na2CO3 (0.354 M) and stirring the mixture at 80 °C overnight. The salt was subsequently removed by filtration; the crude was dried under reduced pressure and dialyzed for 2 days against ultrapure water (SpectraPor dialysis tubes, MWCO 1 kDa). After freeze-drying, PMOXA-OH was obtained as a white solid (72% yield). Both PMOXA-OH and PEOXA-OH were converted to carboxylic acid-terminated analogues by reaction with succinic anhydride. As for PMOXA, PMOXA-OH (1 equiv) and succinic anhydride (10 equiv) were dissolved in dry pyridine and refluxed overnight. The final product PMOXA-COOH was obtained as a white solid after solvent removal, purification via dialysis (SpectraPor, MWCO 1 kDa, 24 h against NaCl solution, 24 h against 10% acetic acid solution, and 24 h against ultrapure water), and final freeze-drying (75% yield). PEOXA-COOH (DP ∼ 100) was synthesized following a similar procedure but using 2-ethyl-2-oxazoline (EOXA) as a monomer and performing the polymerization at 80 °C. Gel permeation chromatography (GPC) (Table S1, Figures S1 and S2) was used to determine the molecular weights of PMOXAs and PEOXAs. Cyclic PMOXA and PEOXA featuring one carboxylic acid function along the macrocycles were synthesized by CROP, using propargyl ptoluenesulfonate as initiator and 2-azidoethylamine as terminating agent, to yield propargyl-PMOXA-NH-N3 and propargyl-PEOXANH-N3 as functional linear precursors to the macrocyclic compounds, both with DP ∼ 100 (75% and 71% yields, respectively). The intramolecular cyclization of these latter species was performed via Cu(I)-catalyzed Huisgen cycloaddition as previously reported.20,24 In particular, 300 mg of propargyl-PMOXA-NH-N3 was dissolved in 200 mL of water, while 75 mg of sodium ascorbate and 60 mg of CuI were dissolved in 1.2 L of water. The two solutions were degassed separately for 1 h under Ar. After this time, the propargyl-PMOXANH-N3 solution was added dropwise to the solution containing CuI/ ascorbate, using a high-precision tubing pump (IPC-N4, ISMATEC, Switzerland) at a rate of 45 μL min−1. After complete addition, the final solution was stirred under Ar for an additional 24 h. The volume was subsequently reduced by a rotavap; the solution was filtered (0.45 μm PTFE Chromafil filters) and finally freeze-dried. The crude product was dissolved in 5 mL of methanol and passed through a column of basic alumina to remove any Cu salt. The solvent was then removed, and the product was redissolved in chloroform, filtered (0.25 μm PTFE Chromafil), and freeze-dried (85% yield). GPC analysis (Table S1, Figures S1 and S2) and FT-IR spectroscopy (Figures S3 and S4) were used to confirm the formation of the cyclic product. cPMOXA-NH and cPEOXA-NH were converted respectively to cPMOXA-COOH and cPEOXA-COOH by reaction with succinic anhydride, following a similar procedure to that described above for their linear counterparts. PLL-g-linear and cyclic PAOXAs (PLL-g-lPAOXA and PLL-gcPAOXA, respectively) were synthesized following a procedure similar to that already reported in the literature.33 Poly-L-lysine hydrobromide (100 mg, 0.48 mmol of NH3+, Mw = 15000−30000 g mol−1, Sigma-Aldrich), the selected side chain l- or cPAOXA-COOH (0.16 mmol, corresponding to 0.33 X/lysine unit), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS, 0.16 mmol), and N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, 1.6 mmol) were dissolved separately in 2 mL of 10 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution (pH 7.4) and mixed in the order given above. The mixture was left under stirring overnight and purified via dialysis (SpetraPor, MWCO 25000 Da) for 2 days. PLL-g-lPAOXAs and PLL-g-cPAOXAs were obtained after freeze-drying as white solids (80% yield on average). The number of side chains per lysine unit (nPAOXA/Lys) was determined by 1H NMR spectroscopy. In particular, after functionalization of the PLL backbone with l- and cPAOXAs, the signals corresponding to both the PLL and the side chains were found in the NMR spectra. As shown in Figures S5−S8, the signal referring to the
surfaces, is of high fundamental relevance, as side chaintopology effects within similar highly branched copolymer species have never been investigated until now, within both graft copolymers and more densely branched bottlebrushes. We specifically synthesized CLPs featuring a positively charged poly-L-lysine (PLL) backbone, which strongly interacts with negatively charged metal oxide surfaces, and cyclic poly(2-alkyl-2-oxazoline)s (PAOXAs) side chains, which can form a dense brush interface. The choice of PAOXAs is highly technologically relevant, as these chemically tailorable polymers44 represent emerging candidates for replacing poly(ethylene glycol) (PEG) and its derivatives in a variety of biomaterials-related applications, including the fabrication of biointerfaces,45−47 tissue engineering constructs,48,49 and therapeutics.50−55 In particular, we focused on PLL-g-cyclic poly(2-methyl-2oxazoline) and PLL-g-cyclic poly(2-ethyl-2-oxazoline) (PLL-gcPMOXA and PLL-g-cPEOXA, respectively) and investigated their assembly process on TiO2 surfaces, chemically identical to those of several biomedical devices, while comparing the deposition mechanism of CLPs presenting linear PMOXA and PEOXA (PLL-g-lPMOXA and PLL-g-lPEOXA). The influence of side-chain topology on CLP assembly kinetics and on the hydration and nanomorphology of the subsequently formed films was investigated through a variety of surface analytics, including quartz crystal microbalance with dissipation (QCM-D), variable-angle spectroscopic ellipsometry (VASE), and atomic force microscopy (AFM), highlighting how CLPs presenting cyclic side chains generate brush layers with significantly altered properties compared to their linear graft-bearing counterparts. The application of PLL-g-cPAOXA films as lubricious biointerfaces was subsequently evaluated through a combination of antifouling and nanotribological tests. Unspecific protein adsorption on CLP films was investigated within serum-rich cell culture media and undiluted full human serum (FHS), while their lubrication properties were compared by lateral force microscopy (LFM), subjecting topologically different PAOXA interfaces to different normal loads and shear velocities. This comprehensive characterization emphasized how CLPs presenting cyclic side chains generate surfaces with enhanced bioinertness with respect to their linear graft-bearing counterparts, and provide extremely low friction when sheared against both a bare inorganic surface and an identical polymer interface. CLPs featuring bioinert, PAOXA-based cyclic segments thus emerge as newly designed functional adsorbates capable of providing unprecedented interfacial properties to a large variety of substrates. These unique traits are chiefly determined by polymer topology effects, which confirm their highly applicative potential when they reveal themselves under the confinement of a grafting surface.
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EXPERIMENTAL SECTION
Synthesis of PLL-g-Linear and Cyclic PAOXA. Hydroxylterminated PMOXA and PEOXA (PMOXA-OH and PEOXA-OH, respectively) with degree of polymerization (DP) of ∼100 were synthesized by cationic ring-opening polymerization (CROP), following the already reported procedures.33 The synthesis of PMOXA-OH is exemplarily reported. 2-Methyl-2-oxazoline (MOXA) (10 g, 118 mmol) was distilled over KOH under Ar atmosphere and subsequently dissolved in 20 mL of dry acetonitrile (ACN). Methyl tosylate (previously distilled over CaH2) (177 μL, B
DOI: 10.1021/acs.macromol.8b02549 Macromolecules XXXX, XXX, XXX−XXX
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dissipation shifts (ΔD), using three different overtones (third, fifth, and seventh) for each sample. Two crystals for each film were used to calculate the mean values of Twet and their standard deviations. The fixed input parameters of the fitting were fluid density (997 kg m−3), layer density (1100 kg m−3), and fluid viscosity (0.009 kg/(m s)). The following parameters were fitted while constrained to physically meaningful boundaries: layer viscosity (0.009−0.05 kg/(m s)), layer shear thickness (104−108 Pa), and layer thickness (10−9−2 × 10−8 m). The obtained values of hydrated mass for PLL-g-PAOXA films were compared to the values of Tdry obtained by VASE (see above), finally obtaining the amount of coupled H2O for each assembly. Atomic Force Microscopy (AFM). High-resolution AFM micrographs in HEPES I buffer solution (pH = 7.4) were acquired by using a Bruker Dimension Icon AFM. In particular, PeakForce Tapping mode was applied to study the nanomorphology of PLL-g-PAOXA and map their nanomechanical and adhesive properties, using a cantilever with spring constant (KN) of ∼0.7 N m−1 (Scanasyst-Fluid +, Bruker AFM probes). A tapping frequency of 2 kHz and a PeakForce set point of 1 nN were used for imaging. Normal and friction force measurements were performed by AFM and lateral force microscopy (LFM), respectively, using a MFP3D AFM (Asylum Research, Oxford Instruments, Santa Barbara, CA) under HEPES I buffer (pH = 7.4). The normal (KN) and the torsional (KT) spring constants of tipless cantilevers (CSC38/tipless/Cr−Au, Mikromash, Bulgaria) were measured by the thermal noise57 and Sader’s method,58 respectively, prior to attachment of a silica colloid to the cantilevers. The colloidal probes were prepared by gluing the silica particle (EKA Chemicals AB, Kromasil R, Sweden) to the end of a tipless cantilever using a home-built micromanipulator. The calibration of the lateral force was performed by using the testprobe method described by Cannara et al.59 The normal and torsional spring constant values along with the diameter of the colloid used for friction force-vs-load (FfL) and friction-vs-scanning velocity measurements are reported in Table S2. Friction force values were obtained by averaging ∼10 measured “friction loops” across each film. The friction loops were acquired by laterally scanning the cantilever on a single line for each applied load over two different positions on the investigated sample.60 A sliding distance of 2 μm and a scanning rate of 0.5 Hz were used for the FfL measurements. Normal force measurements were performed with the same cantilevers used for the friction tests. Approximately 20 force-vsseparation (FS) curves were acquired over two different positions on each sample. A scanning distance of 0.5 μm with the scanning speed of 0.5 μm s−1 was used to obtain the FS curves. The effect of scanning velocity on the measured friction was evaluated within a polymer film-vs-polymer film tribological pair, using a colloidal AFM probe coated with 20 nm thick TiO2 and functionalized with a PLL-g-PAOXA assembly identical to that deposited on the studied substrate. The applied scanning velocities ranged between 20 and 5000 nm s−1. Protein Adsorption Tests. The resistance toward unspecific protein adsorption by the different PLL-g-PAOXA assemblies was evaluated by ex-situ VASE after incubation within different protein media, including 10% human serum in phosphate buffer saline (PBS) (pH = 7.4) and undiluted full hum serum (FHS) (Precinorm U, Roche Diagnostics GmbH, Mannheim, Germany). Initially, PLL-gPAOXA films were incubated in PBS for 5 min to rehydrate, and later on they were immersed in 10% serum and FHS for 30 min. Subsequently, the samples were rinsed with ultrapure water, dried under a stream of N2 and analyzed with VASE. Bare TiO2 substrates were used as positive controls. Three samples for each type of PLL-gPAOXA film were incubated in each protein medium, while one was kept in PBS for the same incubation time to test the intrinsic stability of the assembly at the applied ionic strength. The thickness of the organic layer on each sample was subsequently measured by VASE, according to the previously described procedure.61 The thickness of the physisorbed protein layers was calculated by subtracting the thickness of the copolymer film incubated in PBS to the thickness of PLL-g-PAOXA films subjected to the protein-rich media.
lysine methylene unit next to the terminal amino group was shifted from 3.0 to 3.15 ppm due to amide formation during side-chain grafting. By integration of both peaks (the unmodified −CH2−NH2, denoted as b in Figures S5−S8) and −CH2−N(CO)− (denoted as d in Figures S5−S8), the density of side chains on the PLL backbone was calculated as Id/(Ib + Id). Schematics depicting the synthesis of PLL-g-cPAOXAs and PLL-glPAOXAs are provided in Schemes S1 and S2, respectively. Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR). 1H NMR spectra were recorded by using a Bruker Avance III 700 MHz spectrometer at room temperature and using D2O as solvent. Gel Permeation Chromatography (GPC). Number- and weight-average absolute molecular weights, Mn and Mw, of PAOXAs were determined using an Agilent 1100 size exclusion chromatography (SEC) unit equipped with two PFG linear M columns (PSS) connected in series with an Agilent 1100 VWD/UV detector operated at 290 nm, a DAWN HELEOS 8 multiangle laser light scattering (MALS) detector (Wyatt Technology Europe), and an Optilab T-rEX RI detector from Wyatt. Samples were eluted in hexafluoroisopropanol (HFIP) with 0.02 M K-TFAc at 1 mL min−1 at room temperature. Absolute molecular weights were evaluated with Wyatt ASTRA software and dn/dc values based on our analytical setup (dn/ dcPEOXA = 0.2284 mL/g; dn/dcPMOXA = 0.2498 mL/g). Assembly of PLL-g-PAOXA Films on TiO2. Silicon wafers (SiMat, Landsberg, Germany) were coated by reactive magnetron sputtering (Paul Scherrer Institute, Villigen, Switzerland) with a 17 nm thick TiO2 layer. Prior to functionalization with PLL-g-PAOXA species, the wafers were cut into 1 cm × 1 cm pieces, cleaned by ultrasonication in toluene (2 × 10 min) and isopropanol (2 × 10 min), and dried under a stream of N2. Subsequently, they were treated with UV-ozone (UV Clean Model 135500 from Boekel Industries, Inc.) for 40 min. Freshly cleaned substrates were immersed overnight in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES I), adjusted to pH 7.4 and containing 0.1 mg mL−1 of PLL-g-PAOXA. After copolymer deposition the samples were washed extensively with ultrapure water and finally dried under a stream of N2. Variable Angle Spectroscopic Ellipsometry (VASE). The dry thickness (Tdry) of PLL-g-PAOXA assemblies was evaluated using a M-2000F variable-angle spectroscopic ellipsometer from J.A. Woollam Co. (Lincoln, NE). The values of Ψ and Δ were recorded within a range of wavelengths included between 370 and 1000 nm using focusing lenses at 70° from the surface normal. Fitting of the raw data was performed based on a four-layer model (Si/SiO2/TiO2/Cauchy) using bulk dielectric functions for Si, SiO2, and TiO2. The copolymer layers were analyzed on the basis of the Cauchy model: n = A + Bλ−2, where n is the refractive index, λ is the wavelength, and A and B were assumed to be 1.45 and 0.01, respectively, as values for transparent organic films. All measurements were performed under ambient conditions. Three different substrates were analyzed for each PLL-gPAOXA film, and five points were measured on each sample to calculate the mean values of Tdry and their standard deviations. Quartz Crystal Microbalance with Dissipation (QCM-D). The hydrated thickness of PLL-g-PAOXA films (Twet) was measured by QCM-D using an E4 instrument (Q-Sense AB, Göteborg, Sweden) equipped with dedicated Q-Sense AB software. TiO2-coated crystals (LOT-Oriel AG) with a fundamental resonance frequency of 5 MHz were used as substrates. Before the experiment, the substrates were cleaned by sonication in toluene and 2-propanol and UV-ozone treatment. After cleaning, the crystals were dried under a stream of N2 and immediately used. The crystals were exposed to ultrapure water at 25 °C until a stable baseline was recorded. Later on, ultrapure water was replaced with HEPES I until a new, stable baseline was reached. A 0.1 mg mL−1 HEPES I PLL-g-PAOXA was subsequently injected until complete copolymer adsorption was attained. To determine the stability of the film and eliminate the physisorbed polymers, washing steps were performed with HEPES I and ultrapure water. The values of Twet of each PLL-g-PAOXA film were obtained by applying an extended viscoelastic model,56 fitting the frequency (ΔF) and C
DOI: 10.1021/acs.macromol.8b02549 Macromolecules XXXX, XXX, XXX−XXX
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Scheme 1. PLL-g-PAOXA CLPs Alternatively Featuring Cyclic (a) and Linear Side Chains (b) Assemble on TiO2 Surfaces via Their Positively Charged PLL Backbones Forming Compositionally and Topologically Different PAOXA Brush Interfaces
Table 1. Structural Properties of PLL-g-PAOXA Assemblies CLP film PLL-g-lPMOXA PLL-g-lPEOXA PLL-g-cPMOXA PLL-g-cPEOXA
Tdrya (nm)
σ (nPAOXA nm−2)
Lb (nm)
L/2Rgc
± ± ± ±
0.15 0.15 0.17 0.16
2.8 2.7 2.6 2.7
0.28 0.26 0.38 0.35
2.1 2.9 2.4 3.0
0.1 0.1 0.2 0.1
Twetd (nm)
We (%)
± ± ± ±
85 82 82 79
13.7 16.3 13.3 14.1
1.2 1.5 1.7 1.1
nH2O/PAOXAf 2800 2700 2400 2300
± ± ± ±
150 200 200 250
Measured by VASE. bAverage distance on the surface between PAOXA grafts, calculated using the equation L = (2/√3σ)0.5, where σ is the surface density of PAOXA chains. cDegree of chain overlap; the radius of gyration of each side chain was calculated using the equation Rg = [(Mwη)/(Φ63/2)]1/3, where η is the intrinsic viscosity (46.5 cm3 g−1),63 Φ = 2.68 × 1023 mol−1, and Mw is the weight-average molar mass of the side chains measured by GPC and reported in Table S1. dMeasured by QCM-D. eThe water content within the layers (W) was calculated as (Twet − Tdry)/Twet × 100. fCoupled H2O molecules for PAOXA side chain, calculated from the hydrated polymer mass (QCM-D) compared to the dry mass (VASE), and normalized by σ. a
Statistical analysis was performed with IBM SPSS Statistics (version 24), using a one-way ANOVA, with Tukey’s post hoc test to assess the differences between the adsorbed proteins on linear and cyclic PAOXA brush interfaces. p < 0.05 was noted with an asterisk (∗) while p < 0.01 with two asterisks (∗∗).
chains per unit area (σ), regulates fundamental properties of the obtained brush interfaces, such as their steric stabilization, protein repellence, and lubrication. CLPs presenting linear PMOXA and PEOXA side chains generate brush surfaces with slightly but significantly lower σ with respect to analogous species featuring cyclic grafts. Namely, PAOXA brush density from films of PLL-g-lPMOXA and PLL-g-lPEOXA was 0.15 chains nm−2, while the corresponding cyclic brushes obtained from the surface assembly of PLL-g-cPMOXA and PLL-g-cPEOXA featured σ values of 0.17 and 0.16 chains nm−2, respectively. The formation of denser brushes by PLL-g-cPAOXAs was ascribed to the reduced radius of hydration by cyclic side chains compared to their linear analogues of identical molar mass.18,62 This generates more compact CLPs, which exert a reduced steric hindrance during their assembly on surfaces, ultimately determining the formation of more densely packed films. Interestingly, the effect of polymer topology was less pronounced in the case of surface-interacting CLPs with respect to structurally simpler homopolymer adsorbates, for which a transition from linear to cyclic topology translated into an increment by 40−50% in the density of the subsequently formed brushes.23,24 Noteworthy, all PLL-g-PAOXA films produced brushes characterized by a degree of overlap between neighboring grafts (L/Rg) ≪ 1, confirming the formation of dense brush interfaces irrespective of the topology of CLP side chains. The effects of brush composition and topology on the swelling properties of the generated films were subsequently evaluated by combining VASE and QCM-D data. As summarized in Table 1, PMOXA films were generally more hydrated than PEOXA analogues, the water content (W) of the former ranging from 82 to 85%, with respect to 79−82% by the latter assemblies. This difference in hydration was due to the
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RESULTS AND DISCUSSION PLL-g-PAOXAs assemble on TiO2 surfaces via their positively charged PLL backbones, while generating topologically different PAOXA brushes at the interface (Scheme 1). As was previously demonstrated for similar CLPs based on PEG,25 a relative content of side chains per lysine unit of ∼0.3 (nPAOXA/Lys, defined as y/x in Scheme 1) assures a sufficient concentration of ammonium functions along the PLL to drive the assembly on the TiO2 surface and simultaneously enables the formation of dense PAOXA grafts at the interface. We thus assume a molecular design of surface assemblies of PLL-g-PAOXAs where PLL segments lie down at the substrate, while PAOXA side chains stretch from it, forming a polymer brush layer that mainly constitutes the interface of the films. The composition and topology of CLPs’ side chains determined the structural properties in dry and swollen state of the assemblies, which were analyzed by a combination of VASE and QCM-D (Table 1). Generally, CLPs bearing PMOXA side chains formed slightly thinner films on TiO2 when compared to analogous PEOXA-based layers. This was ascribed to the larger molecular dimensions of PEOXA grafts, generating bulkier copolymer species that assemble on the surface by forming films characterized by higher values of Tdry.33 PAOXA side-chain topology additionally influenced the surface coverage of the different assemblies. This parameter, which is expressed in Table 1 as surface density of PAOXA D
DOI: 10.1021/acs.macromol.8b02549 Macromolecules XXXX, XXX, XXX−XXX
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Figure 1. Assembly of PLL-g-PAOXAs on TiO2 surfaces was recorded in situ by QCM-D by measuring the variations of ΔF and ΔD for (a) PLL-glPMOXA, (b) PLL-g-cPMOXA, (d) PLL-g-lPEOXA, and (e) PLL-g-cPEOXA. The values of ΔF and ΔD for each copolymer type were subsequently fitted with an extended viscoelastic model56 yielding the variation of hydrated polymer mass on the TiO2 sensors for (c) PLL-gPMOXAs and (f) PLL-g-PEOXAs (see Experimental Section for details).
Figure 2. PeakForce AFM height micrographs recorded in HEPES I on assemblies of PLL-g-lPMOXA (a), PLL-g-cPMOXA (b), PLL-g-lPEOXA (c), and PLL-g-cPEOXA (d). Representative FS profiles obtained during scanning were reported in (e) for PLL-g-lPMOXA, in (f) for PLL-gcPMOXA, in (g) for PLL-g-lPEOXA, and in (h) for PLL-g-cPEOXA. Distribution of adhesion force recorded on PLL-g-lPEOXA (light blue histogram) and PLL-g-cPEOXA (dark blue histogram) (i). Schematics depicting the proposed mechanism of interaction between the retracting Si3N4 probe and topologically different PEOXA grafts are also reported as insets in (i).
function of adsorption time on TiO2-coated sensors (Figure 1). The adsorption of PLL-g-PAOXAs featuring linear side chains followed relatively fast kinetics, as witnessed by the sharp decrease of ΔF, which directly correlated to a comparatively fast increment in adsorbed polymer mass on the sensors (Figures 1a and 1d for PLL-g-lPMOXA and PLL-glPEOXA, respectively). Following ∼20 min of adsorption, the values of ΔF reached a plateau, suggesting that after a relatively short incubation time the surface was already saturated by assembled PLL-g-PAOXAs. In contrast, comblike polymers bearing cyclic side chains displayed much more gradual adsorption profiles, ΔF progressively decreasing across the
more amphiphilic character of PEOXA-bearing assemblies, if compared to the more hydrophilic PMOXA-based analogues. In addition, cyclic brush layers showed a comparatively lower hydration with respect to their linear counterparts, presumably due to the more densely packed nature of the former assemblies. In particular, the amount of water molecules coupled to each PAOXA graft (nH2O/PAOXA) decreased from 2800−2700 for PLL-g-lPAOXAs to 2400−2300 for PLLg-cPAOXA layers. To further investigate the effect of side-chain topology on the assembly process of PLL-g-PAOXAs, we monitored the formation of the different CLP films in-situ by QCM-D, recording frequency (ΔF) and dissipation shifts (ΔD) as a E
DOI: 10.1021/acs.macromol.8b02549 Macromolecules XXXX, XXX, XXX−XXX
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Figure 3. (a) Representative FS profiles recorded in HEPES I medium on the different comblike polymer films by colloidal probe AFM. (b) FfL profiles obtained by LFM on PLL-g-PAOXA films by using a silica colloidal probe. The values of FfL previously reported by Divandari et al.23 are also included for comparison. (c) Friction force as a function of scanning velocity recorded by LFM, using a TiO2-coated colloidal probe alternatively bearing a PLL-g-lPMOXA and PLL-g-cPMOXA layer, which was sheared at a normal load of 45 nN against an identical film deposited on a flat TiO2 substrate.
Also in the case of CLP adsorbates the topology of the cyclic side chains was thus demonstrated to substantially alter the interfacial structure of the subsequently formed assemblies. PeakForce AFM further enabled to monitor the interaction between the scanning AFM Si3N4 probe and the different films by recording force vs separation profiles (FS), which highlight their nanomechanical and adhesive properties. As reported in Figures 2e and 2f, both PLL-g-lPMOXA and PLL-g-cPMOXA assemblies showed repulsive interactions toward the AFM probe due to the highly swollen nature and hydrophilicity of PMOXA grafts (Table 1). In contrast, significant adhesion was recorded along the retracting FS profiles on PLL-g-lPEOXA and PLL-g-cPEOXA (Figures 2g and 2h, respectively), indicating the occurrence of attractive forces between the more amphiphilic and less swollen PEOXA chains and the withdrawing probe, presumably through van der Waals interactions.33 As it was observed while comparing the swelling properties of films alternatively produced from PEOXA and PMOXA-bearing CLPs (Table 1), the presence of an ethyl group within the side chains of PEOXA signifincantly decreased its hydration and substantially altered the interfacial properties of the subsequently generated assemblies. Interestingly, the recorded adhesion force was considerably lower on cyclic PEOXA brushes with respect to chemically identical films exposing linear grafts (Figure 2i). As the different strength of interaction with the AFM probe was necessarily correlated to the grafted-chain topology, we assumed that cyclic grafts adhered more weakly on the AFM tip and could be pulled out from their equilibrium configuration to a lower extent when compared to their linear counterparts. Linear PEOXA brushes are instead sterically less hindered than their cyclic analogues and could presumably form multiple adhesive sites on the inorganic surface of the AFM probe, resulting in higher adhesion forces (insets in Figure 2i). The nanomechanical properties of the PLL-g-PAOXA films analyzed by PeakForce AFM were confirmed by applying colloidal probe AFM, recording FS profiles with a silica sphere of 30 μm in diameter attached to an AFM cantilever. Also in this case, PLL-g-PMOXA films showed repulsive interactions with the colloidal probe, irrespective of their side-chain topology, whereas the FS profiles recorded on PLL-g-
whole incubation time, without reaching an evident plateau (Figures 1b and 1e for PLL-g-cPMOXA and PLL-g-cPEOXA, respectively). These differences in adsorption mechanism were confirmed by comparing the variation of hydrated polymer mass adsorbed on the QCM-D sensors during incubation in the different PLLg-PAOXA solutions (Figures 1c and 1f for PLL-g-PMOXAs and PLL-g-PEOXAs, respectively). Both PLL-g-lPMOXA and PLL-g-lPEOXA reached a nearly constant value of adsorbed mass within few minutes from the exposure of the sensors to the polymer solutions, whereas PLL-g-cPMOXA and PLL-gcPEOXA showed an initial fast adsorption, followed by slower although progressive increment in physisorbed polymer mass. As the affinity toward the negatively charged TiO2 surface is solely determined by the charge density along the PLL backbone, which is expected to be constant for PLL-gPAOXAs presenting the same nPAOXA/Lys, the different adsorption kinetics characteristic of CLP presenting cyclic side chains was necessarily ascribed to their overall more compact dimensions, determined by the reduced hydration radius of the cyclic grafts compared to their linear counterparts. Similarly reactive but less bulky CLPs thus showed an initial fast adsorption, leading to films characterized by incomplete coverage, which was followed by a slower rearrangement with a concomitant passivation of areas on the TiO2 surface still exposed to the medium. This process ultimately led to denser CLP films by PLL-g-cPMOXA and PLL-g-cPEOXA after overnight incubation, as recorded by VASE and reported in Table 1. The nanomorphology of the different PLL-g-PAOXA assemblies and their nanomechanical properties were subsequently investigated by PeakForce Tapping AFM performed in aqueous medium (Experimental Section). As reported in Figure 2, PLL-g-cPMOXA and PLL-g-cPEOXA generated assemblies with a smoother and morphologically more uniform interface, if compared to those produced by their linear sidechain-bearing analogues. The formation of smoother nanomorphologies by cyclic adsorbates compared to their linear counterparts was already reported in the case of assemblies of topologically different homopolymers,23,64,65 and it was ascribed to the intrinsic absence of dangling linear chains. F
DOI: 10.1021/acs.macromol.8b02549 Macromolecules XXXX, XXX, XXX−XXX
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for cyclic polyacrylamide chains in bulk when confined on a flat macroscopic surface.68 This distinctive structure generates a highly lubricious layer that significantly reduces friction when slid past the colloidal probe. When the shearing surface is replaced with PLL-g-PMOXA films deposited on TiO2-coated probes, two identical CLP films are sheared against one another (as schematized in Figure 4b), and the effect of side-chain topology on their lubrication properties is further highlighted. The recorded friction force between PLL-g-lPMOXA and PLL-g-cPMOXA films progressively increases with the scanning velocity following a sublinear trend, as theoretically described69,70 and experimentally proven71 for two brush surfaces sheared within a good solvent (Figure 3c). However, friction forces recorded between cyclic brushes are significantly lower compared to those observed between their linear counterparts, within the whole range of scanning rates tested. It is remarkable that the observed dependency of friction on scanning velocity between linear and cyclic grafts is qualitatively in good agreement with the recent results of molecular dynamic simulations by Erbas and Paturej.72 Besides the formation of a smooth interface between sheared cyclic grafts, the remarkably low friction recorded during their reciprocating sliding could be ascribed to a substantial reduction of interpenetration between the opposing cyclic brushes with respect to their linear analogues. In particular, penetration of a grafted chain within a topologically identical, opposing brush is more entropically unfavorable in the case of cyclic adsorbates compared to the corresponding linear, as the former should fold and double up to interdigitate with the brush-bearing countersurface.73 The consequent reduction in brush overlapping under shear leads to a decrease in the number of collisions between grafted chains, thus generating lower frictional forces.72 Because a highly lubricious character by polymer brush layers is typically associated with a marked repulsion toward unspecific protein contamination,61 we finally tested the antifouling properties of PLL-g-PAOXA assemblies within complex protein media, including 10% serum solutions (Figure 5a), which are analogous to protein-rich cell culture media, and undiluted full human serum (Figure 5b).
PEOXA assemblies highlighted a significant adhesion, which was more marked in the case of PLL-g-lPEOXA (Figure 3a). To determine the effect of side-chain topology on the nanotribological properties of the assembled films, lateral force microscopy (LFM) was subsequently applied with analogous AFM colloidal probes, recording friction force vs applied load profiles (FfL).60 As shown in Figure 3b, when brush surfaces from PLL-g-lPEOXA are sheared against the bare silica colloid (within a tribological system depicted in Figure 4a), the
Figure 4. LFM was applied to investigate the nanotribological properties of PLL-g-PAOXAs featuring topologically different side chains. CLP assemblies were sheared against a bare silica colloid to record FfL profiles (a). Alternatively, AFM colloidal probes bearing CLP assemblies identical to those deposited on the flat TiO2 surface were employed to investigate the effect of scanning velocity on the measured friction force (b).
highest friction among the different assemblies studied was recorded, probably due to the amphiphilic character and reduced swelling by linear PEOXA grafts, providing a coefficient of friction (μ) of 0.37. The replacement of linear grafts with cyclic PEOXA analogues determined a substantial reduction in friction, the FfL profiles recorded on PLL-g-cPEOXA films corresponding to a μ of 0.22. The FfL data obtained from PEOXA-bearing CLP substantially agreed with the values of friction previously recorded by Divandari et al.,23 investigating the nanotribological properties of linear and cyclic PEOXA homopolymer brushes (dotted profiles in Figure 3b). However, in the latter case, the grafting density of PEOXA brushes was significantly higher than that obtained from CLP assembly, and thus the absolute values of friction force were generally lower. A similar although more marked effect was observed by comparing the nanotribological properties of PLL-g-lPMOXA and PLL-g-cPMOXA assemblies. Linear PMOXA grafts showed a further improvement in lubrication compared to the more amphiphilic linear and cyclic PEOXA grafts, providing a μ = 0.14. However, the reduction in friction attained by shearing cyclic PMOXA interfaces was even more remarkable. Namely, friction recorded on PLL-g-cPMOXA was extremely low across the whole range of applied loads tested, reaching a μ of 0.006a value that is among the lowest ever measured by AFM techniques on nonionic brush surfaces and that approximates the highly lubricious behavior typically observed for polyelectrolyte films in water.66,67 The exceptionally low friction recorded on PLL-g-cPMOXA was ascribed to the molecularly smooth interface generated by cyclic grafts compressed under shear by the colloidal AFM probe, coupled to their high hydration. As depicted in Figure 4a, in the absence of dangling linear chains at the interface, cPMOXA grafts flatten their equilibrium configuration when a shearing load is applied, in a similar way to what was described
Figure 5. Protein resistance properties of PLL-g-PAOXA assemblies are evaluated by VASE after 1 h of incubation in 10% serum PBS solutions (a) and undiluted, full human serum (FHS). p < 0.05 was noted with (∗) while p < 0.01 with (∗∗). G
DOI: 10.1021/acs.macromol.8b02549 Macromolecules XXXX, XXX, XXX−XXX
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In agreement with our previous study focusing on the protein repellent behavior by different linear PAOXA brushes, PMOXA interfaces showed a lower amount of adsorbed proteins compared to PEOXA analogues.33 However, a significant variation in biopassivity was observed by comparing CLP films presenting different side-chain topologies. Specifically, both PLL-g-cPEOXA and PLL-g-cPMOXA layers showed a significant reduction in adsorbed proteins compared to the corresponding linear grafts-bearing CLP films. Moreover, it is remarkable that assemblies of PLL-g-cPMOXA demonstrated a nearly quantitative bioinertness toward serum-containing media, assuring an adsorbed protein thickness always
