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Biocompatible Nanocoatings of Fluorinated Polyphosphazenes through Aqueous Assembly Victor Selin, Victoria Albright, John F. Ankner, Alexander Marin, Alexander K. Andrianov, and Svetlana A. Sukhishvili ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02072 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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ACS Applied Materials & Interfaces

Biocompatible Nanocoatings of Fluorinated Polyphosphazenes through Aqueous Assembly Victor Selin1, Victoria Albright1, John F. Ankner2, Alexander Marin3, Alexander K. Andrianov3*, and Svetlana A. Sukhishvili1* 1

Department of Materials Science & Engineering, Texas A&M University, College Station, Texas 77843, USA

2 3

Spallation Neutron Source, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA

Institute for Bioscience and Biotechnology Research, University of Maryland, Gaithersburg, MD, USA

Abstract Nonionic fluorinated polyphosphazenes, such as poly[bis(trifluoroethoxy)phosphazene] (PTFEP), display superb biocompatibility, yet their deposition to surfaces has been limited to solution casting from organic solvents or thermal molding. Herein, hydrophobic coatings of fluorinated polyphosphazenes are demonstrated through controlled deposition of ionic fluorinated polyphosphazenes (iFPs) from aqueous solutions using the layer-by-layer (LbL) technique. Specifically, the assemblies included poly[(carboxylatophenoxy)(trifluoroethoxy) phosphazenes] with varied content of fluorine atoms as iFPs (or poly[bis(carboxyphenoxy) phosphazene] (PCPP) as a control nonfluorinated polyphosphazene) and a variety of polycations. Hydrophobic interactions largely contributed to the formation of LbL films of iFPs with polycations, leading to linear growth and extremely low water uptake. Hydrophobicity-enhanced ionic pairing within iFP/BPEI assemblies gave rise to large-amplitude oscillations in surface wettability as a function of capping layer, which were the largest for the most fluorinated iFP, while control PCPP/polycation systems remained hydrophilic regardless of the film top layer. Neutron reflectometry (NR) studies indicated superior layering and persistence of such layering in salt solution for iFP/BPEI films as compared to control PCPP/polycation systems. Hydrophobicity of iFP-capped LbL coatings could be further enhanced by using a highly porous polyester surgical felt rather than planar substrates for film deposition. Importantly, iFP/polycation coatings displayed biocompatibility which was similar to or superior to that of solution-cast coatings of a clinically-validated material – PTFEP, as demonstrated by the hemolysis of the whole blood and protein adsorption studies. Keywords: polyphosphazenes, layer-by-layer films, polyelectrolytes, biocompatibility, surface wettability

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INTRODUCTION Current development of advanced materials for implants, coronary stents, and catheters often involves both organic and inorganic components.1-4 Such hybrid materials can advantageously combine, for example, the excellent mechanical properties and durability of inorganic materials (such as titanium and ceramics) with the capability of polymers to self-heal or serve as a platform for drug delivery and control cellular proliferation.5 Traditionally, polymers are deposited on surfaces of biomaterials via heat molding or solution casting,6-8 but controlling the uniformity, thickness, and functionality of the coating using these methods is challenging.9 In contrast, the layer-by-layer (LbL) deposition technique enables coating substrates of a variety of shapes and surface chemistries with nanoscopically structured films.10 The LbL technique has been widely used to create surface coatings for functionalization of biomedical devices to control cellular adhesion and localized delivery of bioactive molecules.6,11,12 Polyphosphazenes (PPZs) − “hybrid” polymers, with an inorganic backbone of alternating phosphorus and nitrogen atoms and organic side groups, hold significant potential as biomaterials.13-14 These synthetic macromolecules are distinct due to the combination of unprecedented backbone flexibility and facile tunability of functionality through the diverse chemistry of the organic substituents.13-15 In particular, films of nonionic fluorinated PPZs (FPs) prepared by casting from organic solvents provided surfaces with added hydrophobicity16 and demonstrated exceptional biocompatibility in preclinical and clinical settings.17-21 Yet the intrinsic hydrophobicity of this polymer and lack of functional groups for binding with partner macromolecules precludes incorporation of nonionic FPs within nanostructured films using the LbL technique. Nonetheless, non-fluorinated ionic PPZs were previously deposited via electrostatic22 or hydrogen bonding LbL assembly,23 yielding films with ionic conductivity an order of magnitude higher than that achievable with assemblies of traditional non-PPZ polyelectrolytes. Here, for the first time, we report on controlled LbL assembly of ionic fluorinated PPZs (iFP), explore the structure of the resultant LbL films, and demonstrate their capability to control surface hydrophobicity and interactions with biological milieu. This approach is enabled by iFPs 2 ACS Paragon Plus Environment

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designed

to

contain

both

fluorinated

and

carboxylic

acid

moieties,

i.e.

poly[(trifluoroethoxy)(carboxylatophenoxy)phosphazenes].24 Remarkably, these iFPs maintained their solubility either in aqueous solutions or in alcohol-water mixtures even when the content of hydrophobic fluorinated groups was as high as 86 %,24 which made them attractive candidates to explore with the LbL coating technique. Since the interactions of polymer coatings with biological environments are largely controlled by the film surface energy and swelling, this work first focused on studies of film deposition, wettability, and water uptake, and later correlated these properties with the film internal structure. The main physico-chemical characteristics of LbL films are related to their film growth. Two distinct regimes are usually recognized, i.e. linear growth – when the thickness of a film increases linearly with the number of deposited layers – and non-linear growth characterized by a rapid increase in film thickness with the polymer mass deposited per layer continuously increasing with layer number. Different film growth regimes occur due to varied strength of interpolymer binding25-26 and diffusivity of the polymer components.27-28 It is generally accepted that strongly paired polymers demonstrate linear growth, layered film structure, and a low degree of water uptake,29-33 while weakly bound polyelectrolytes yield highly swollen, intermixed assemblies.29 Such differences in strength in polymer-polymer interactions are critical for achieving controlled film surface characteristics, such as wetting and interactions with proteins and/or whole blood. This work aims to integrate the outstanding hemocompatibility of fluorinated polyphosphazenes6, 17, 20, 34 with the versatility and control of the film layering afforded by the LbL technique. While trifluoroethoxy groups of iFPs support hemocompatibility of the coatings, its carboxylic acid functionalities enable water-solubility and assembly of these polyelectrolytes. Films of iFPs with varied content of fluorinated moieties were assembled from aqueous solutions, and the effects of hydrophobicity, charge density, and binding partner on film growth were explored. The film growth, hydrophobicity, and swelling could be all tuned by the fluorination degree of iFPs, and highly hydrophobic surfaces were achieved through a fully aqueous-based LbL deposition. The nanocoatings of iFPs demonstrated exceptional blood compatibility that was comparable with solution cast films of a clinically-validated material – PTFEP. Importantly, iFP-containing LbL films could be deposited on several biomedically relevant substrates and demonstrated consistent surface characteristics. 3 ACS Paragon Plus Environment


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Experimental Section Materials. Hydrogenated and fully deuterated dimethyl sulfate (d6) as well as all solvents were obtained from Sigma-Aldrich. Ultrapure Milli-Q water (Millipore) with a resistivity of 18.2 MΩ/cm was used in all experiments. Rhodamine 6G was purchased from Alfa Aesar. Deuterated poly(2-(dimethylamino)ethyl methacrylate) (dPDMAEMA) homopolymer with Mw 90 kDa and PDI of 1.8 was purchased from Polymer Source Inc. Rabbit blood in sodium citrate was purchased from Rockland Immunochemicals, Inc. Anti-human serum albumin antibody (Biotin) and streptavidin-HRP conjugate were purchased from Abcam. Hemoglobin, lyophilized powder, and human plasma were purchased from Sigma. Phosphate buffered saline (PBS, pH 7.4) was purchased from Thermo-Fisher Scientific. All other chemicals were purchased from SigmaAldrich and used without further purification. Silicon wafer substrates (111 orientation) were purchased from University Wafer, Inc. Titanium foil sheets of thickness 0.25 mm with 99.7% trace metals basis were purchased from Sigma-Aldrich. Polyester surgical felt was purchased from Bard Peripheral Vascular.

Polymer Synthesis and Characterization Polyanions:

Two

types

of

ionic

FPs

(iFPs)

of

poly[(trifluoroethoxy)(carboxylatophenoxy)phosphazene] family, containing 60 and 86 molar% of trifluoroethoxy groups and abbreviated as FP60 and FP86, respectively, as well as nonfluorinated

polyphosphazene

polyelectrolyte

-

poly[di(carboxylatophenoxy)phosphazene]

(PCPP) were synthesized as reported previously.24,35 Non-ionic fluorinated homopolymer, poly[bis(trifluoroethoxy)phosphazene]

(PTFEP),

was

synthesized

by

reacting

poly(dichlorophosphazene), which was obtained by ring opening polymerization of hexachlorocyclotriphosphazene as described previously,36 with sodium 2,2,2-trifluoroethoxide.13 Mws were determined by gel permeation chromatography (GPC) in PBS (pH 7.4) or N,Ndimethylacetamide containing 0.1% tetra-n-butyl ammonium bromide and were as follows: 800 kDa for PCPP (multi-angle laser light scattering), 246 kDa for FP60, 295 kDa for FP86, and 1,100 kDa for PTFEP (polystyrene standards). Polycations:

Hydrogenated

poly(2-(dimethylamino)ethyl

methacrylate)

(hPDMAEMA)

homopolymer was synthesized by atom transfer radical polymerization (ATRP) as previously 4 ACS Paragon Plus Environment

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described.37 GPC analysis of hPDMAEMA was performed in DMF with polystyrene (PS) standards. The Mw and PDI of the homopolymer were 90 kDa and 1.10, respectively, as determined by GPC. Quaternization of hPDMAEMA to obtain a 100% quaternized polycation with a molecular weight of 95 kDa, abbreviated here as QPC, was carried out at room temperature. To synthesize QPC, hPDMAEMA was dissolved in a mixture of ethanol/benzene (v:v = 3:1), and a stoichiometric amount of hydrogenated dimethyl sulfate was added to the solution. The mixture was stirred at room temperature overnight. The precipitated product was washed with acetone three times and dried under vacuum overnight. A similar procedure was carried out to synthesize deuterated quaternized polycation (dQPC). To that end, dPDMAEMA was treated with fully deuterated rather than hydrogenated dimethyl sulfate. Branched polyethyleneimine (BPEI) with a weight-average molecular weight (Mw) of 25 kDa and polydispersity index (PDI) of 2.5 and poly-L-lysine (PLL) with Mw of 4-15 kDa were purchased from Sigma-Aldrich.

Multilayer Buildup. LbL films were assembled on silicon wafers, titanium foil, or polyester surgical felt. Prior to film deposition, silicon wafers were cleaned as described elsewhere.38 Similarly, titanium foil was cut into 1 cm2 pieces and cleaned with the same procedure as silicon wafers. Polyester surgical felt was treated with oxygen plasma for 10 minutes. All substrates were primed with a monolayer of BPEI, which was allowed to adsorb from 0.2 mg/ml solution at pH 9 for 15 min. LbL films were then deposited by dip- or spin-coating from 0.2 mg/mL polyelectrolyte solutions in 0.01 M phosphate buffer at pH 7.4. For dip coating, substrates were sequentially dipped in PPZs and polycation solutions for 5 min with a washing step in between, which consisted of immersion twice in 0.01 M phosphate buffer solutions at pH 7.4 for 2 min. For construction of FP86 LbL films, all deposition solutions contained 10% of ethanol. For spincoating, a Laurel WS-650-23NNP/UD3/UD3B spin coater was used with a 40 s per step deposition cycle and a rotational speed of 3000 rpm. Each deposition step was followed by washing the substrate with 0.01 M phosphate buffer for 40 s at the same rotational speed. To provide contrast for neutron scattering measurements, deuterated QPC, dQPC, was used to produce a ten-bilayer stack enriched with the deuterated material dQPC/PPZ using the spin-coating method. This stack was deposited on top of a stack of the hydrogenated material composed of ten bilayers of QPC/PPZs. The final sample design for PCPP and FP60 containing 5 ACS Paragon Plus Environment


films

was

Si/SiO2/BPEI/(PCPP/QPC)10/(PCPP/dQPC)10

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and

Si/SiO2/BPEI/(FP60/QPC)10/(FP60/dQPC)10, respectively, where each ten-bilayer stack was denoted as PCPPH, PCPPD, FP60H and FP60D. Coating Wettability. Contact angle measurements were performed with LbL films deposited onto silicon wafers, titanium foil, or polyester surgical felt. The contact angles formed by a droplet of buffer solution at pH 7.4 were measured using a KSV Instruments Ltd. instrument equipped with an ImagineSource camera and OneAttension software. The contact angle values were averaged over 10 measurements.

To visualize coating wettability on porous polyester surgical felt, a small amount of Rhodamine 6G was dissolved in Milli-Q water, ~0.05 mg/mL, to enhance droplet visibility. 200 µL of Rhodamine 6G solution was added on top of surgical felt substrates coated with LbL films terminated with either BPEI or PPZ. Coatings were imaged after a 30-s waiting period.

Spectroscopic Ellipsometry Measurements. Thicknesses and optical constants in both dry and swollen states of LbL films were characterized using a variable angle spectroscopic ellipsometer (VASE, M-2000 UV−visible−NIR [240−1700 nm] J. A. Woollam Co., Inc., Lincoln, NE, USA) equipped with a temperature-controlled liquid cell. Prior to deposition of LbL films, the thickness of the oxide layer on the silicon substrate was measured. Dry measurements were carried out at three angles of incidence: 45°, 55°, and 65°.To fit the ellipsometric data from dry films, a three-layer model was used, in which the first two layers representing the silicon substrate and its oxide layer, and the third layer representing the LbL film. The polymer layer was treated as a Cauchy material of thickness d, having a wavelength-dependent refractive index n(λ) = A + B/λ2 + C/λ3, where A, B and C are fitting coefficients, and λ is the wavelength. The film extinction coefficient was assumed to be negligible (k = 0). Thickness d and the three coefficients A, B and C were fitted simultaneously. For measurements in the liquid cell, the cell geometry dictated that the angle of incidence be 75°. In all experiments, the temperature was set to 25 °C. To avoid effects of absorption in the ultraviolet and near-infrared light region by the buffer solution, the working wavelength band was set to 370.5 – 999 nm. For in situ ellipsometry experiments, a silicon wafer with a pre6 ACS Paragon Plus Environment

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deposited film of known dry thickness was placed into a liquid cell. The cell was then filled with 0.01M buffer at pH 7.4 and a thickness measurement was taken after a waiting period of 10 min. The measurements were finalized after a constant wet thickness was reached and then by removing the sample from the cell, drying with nitrogen flow, and measuring the dry thickness again. To fit the ellipsometric data for the in situ measurements, a four-layer model was used. An additional layer represents the semi-infinite buffer solution and was also treated as a transparent Cauchy medium, with a wavelength-dependent refractive index nbuf(λ) = Abuf + Bbuf/(λ)2 + Cbuf/(λ) 3, where Abuf, Bbuf and Cbuf are fitting coefficients, and λ is the wavelength. For the buffer solutions, Abuf, Bbuf and Cbuf were determined prior to in situ experiments by measuring nbuf(λ) for a bare, clean silicon wafer installed in the liquid cell containing 0.01 M phosphate buffer at pH 7.4. After completion of the in situ measurements, the dry thicknesses of the films were measured again to assure that the thicknesses used in the swelling experiments were consistent with the independently measured dry film thicknesses obtained in the film growth experiments. In all experiments, the coefficients A, B, C were consistent within 5%.

Neutron Reflectometry (NR) Measurements. NR measurements were first performed with dry LbL films deposited at the surface of silicon wafers from solutions at low ionic strength (0.01 M phosphate buffer at pH 7.4). After these measurements, the samples were transferred to a 0.4 M NaCl solution in phosphate buffer at pH 7.4 for 10 min to stimulate chain diffusion, washed with low-ionic-strength buffer, dried in nitrogen flow, and measured again. Samples prepared for NR studies were assembled using a two-stack design. For each stack in the fitting model, the layers’ thickness, scattering length density (SLDs), and interfacial roughnesses were varied. NR measurements were performed at the Spallation Neutron Source Liquids Reflectometer (SNS-LR) at the Oak Ridge National Laboratory (ORNL). The reflectivity data were collected using a sequence of 3.4-Å-wide continuous wavelength bands (selected from 2.55 Å < λ < 16.7 Å) and incident angles (ranging over 0.6° < θ < 2.34°). The momentum transfer, Q = (4π sin θ/λ), was varied over a range of 0.008 Å–1 < Q < 0.193 Å–1. Reflectivity curves were assembled by combining seven different wavelength and angle data sets together, maintaining a constant relative instrumental resolution of δQ/Q = 0.023 by varying the incidentbeam apertures. Scattering densities within hydrogenated and deuterated stacks were averaged 7 ACS Paragon Plus Environment


over the 10 constituent bilayers, with each stack exhibiting its characteristic thickness, scatteringlength density, and interlayer roughness. Those characteristic parameters were adjusted until the reflectivity curve was best fitted (minimized χ2). Blood Compatibility Studies. Hemolysis tests of multilayer coatings were performed by incubating samples with dilute whole rabbit blood at 37° C for four hours.39 Prior to experiments, rabbit blood in sodium citrate (Rockland Immunochemicals, Inc., Limerick, PA 19468) was diluted 50 times with PBS to a hemoglobin concentration of 2 mg/mL. The test samples (220 mm2 silicon wafers coated with PPZ-containing films) were put into 10-mL syringes, to which 0.5 mL of diluted blood was added, then the plunger was reinstalled and the air was removed. The syringes were capped and incubated at 37° C using an InnovaTM 2100 shaker platform. The suspensions were then centrifuged for 5 minutes (14000 rpm, Microfuge® I8 Centrifuge) and the concentration of hemoglobin in supernatant was analyzed by UV-VIS spectrophotometry using Multiscan Spectrum microplate spectrophotometer (ThermoFisher Scientific, Waltham, MA).39 Hemoglobin, lyophilized powder (Sigma, Milwaukee, WI) was used to prepare a calibration curve. All samples were analyzed in triplicate. The hemolysis, % was reported as a difference between the % of free hemoglobin in the blood before and after incubation with the tested material.

Selective Protein Adsorption Studies. Selective human serum albumin (HSA) adsorption tests were performed using human plasma and enzyme-linked immunosorbent assay (ELISA).6 The coated samples were placed in a 24-well plate and 0.25 mL phosphate buffered saline (PBS, pH 7.4), was added to each sample-containing well. After 10 minutes, 0.25 mL of 20 % (v/v) human plasma (Sigma, Milwaukee, WI) was added to each well and then samples were incubated for 30 minutes at ambient temperature. Next, human plasma was removed and the plate was washed with PBS. To prevent non-specific interactions, 0.5 mL of a blocking buffer [1 wt % bovine serum albumin (BSA) in PBS] was added to each well. After 30 minutes, the plate was rinsed with PBS. Then 0.5 mL of 0.5 µg/mL anti-human serum albumin antibody (Biotin) (Abcam, Cambridge, MA, ab27632) in PBS containing 0.1% BSA was added to each well, samples were incubated for 15 minutes at ambient temperature, and then rinsed with PBS. Next, 0.5 mL of 0.5 µg/mL streptavidin-HRP conjugate (Abcam, Cambridge, MA, ab7403) in PBS containing 0.1 wt % BSA was added to each well, samples were incubated for 15 minutes and rinsed with PBS 8 ACS Paragon Plus Environment

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again. Finally, 0.4 mL of PBS was added to each well, followed by the addition of 100 µL of TMB peroxidase EIA substrate kit solution (Bio-Rad, Hercules, CA). After 20 minutes, the reaction was stopped by adding 100 µL of 1 M sulfuric acid, and optical density at 450 nm was measured using a Multiscan Spectrum microplate spectrophotometer (ThermoFisher Scientific, Waltham, MA) to determine the amount of HSA adsorbed. The irreversible part of adsorbed HSA was determined by rinsing plasma-treated samples with 0.5 wt% solution of sodium dodecyl sulfate in PBS6 and determining the amount of HSA as described above.

RESULTS AND DISCUSSION Film growth with various polyphosphazene components. To compare the effects of polyphosphazene (PPZ) chain hydrophobicity and chemical makeup on film growth, a series of LbL films with various polycations and PPZ partners were studied (Scheme 1).

Scheme 1. Chemical structures of the components and schematic representation of the LbL assembly. Fig. 1A demonstrates deposition of the LbL assemblies of an ionic PPZ containing 60% of fluorinated side groups− FP60 − with a set of various polycations. The growth was linear with a constant increment in the amount deposited per bilayer (~ 4.8 nm per bilayer) for all FP60 assemblies with various polycations (BPEI, PLL, and QPC). Such deposition indicated strong binding between assembled macromolecules. Fig. 1B compares these assemblies with films of a more

common

water-soluble

PPZ,

which

lacks

fluorinated

functionality,

i.e.

poly[di(carboxyphenoxy)phosphazene] (PCPP). In the case of PCPP, thicker films are formed with all electrostatic partners, with the thickest films (exceeding 100 nm in just 6 bilayers) for 9 ACS Paragon Plus Environment


the PCPP/QPC system, in which electrostatic polymer-polymer pairing energy was significantly lowered by sterical hinderance at the quaternary ammonium group of QPC.40 The strikingly different modes of film growth with two different types of polyphosphazenes − iFPs and PCPP − point to different strength of ionic pairing29, 41 and to the importance of hydrophobic interactions in LbL films growth.42-43 To further study the effect of the additional hydrophobic interactions brought in by the

Fig. 1. (A) Dry thicknesses of LbL films of FP60 or PCPP and a set of polycations (A and B, respectively) as a function of bilayer number; (C) Dry thicknesses of LbL films of BPEI and set of PPZs with various degrees of fluorination as a function of bilayer number; (D) Comparison of the bilayer thicknesses for various LbL films deposited via either the dip- or spin-assisted technique. fluorinated moieties, PPZs with varied content of trifluoroethoxy groups were assembled within LbL films with the same polycation. Fig. 1C compares film growth for a non-fluorinated PPZ 10 ACS Paragon Plus Environment

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(PCPP) as well as fluorinated FP60 and FP86 with BPEI. For the fluorinated polyelectrolytes, the rates of the film thickness increase were similar (3.9 to 3.8 nm per bilayer for FP60 and FP86, respectively), but twice thicker films were formed with non-fluorinated PCPP. For both FP60 and FP86, which had similar molecular weight (see Experimental section), the introduction of fluorinated groups increased the polymer-polymer binding strength due to additional hydrophobic interactions, leading to thinner, linearly depositing films. The differences in the macromolecular binding strength of fluorinated and non-fluorinated PPZs became even more obvious in the experiments in which spin-assisted rather than dip-assisted assembly was used. Fig. 1D shows that the action of the centrifugal force during spin-assisted deposition (Fig. S1) greatly affected the outcome of assembly in the case of the weakly bound PCPP. Specifically, the polymer chains became flattened, resulting in thinner LbL films. Previously, our group reported a similar result while studying a different LbL system of nonfluorinated polyelectrolytes.32 In contrast, FP60-containing films, in which polymer chains are initially much stronger associated, demonstrated only a slight decrease in bilayer thickness.

Film swelling and surface wettability. The modes of LbL film growth are strongly coupled with the capability of the film to uptake water and to controllably present polyelectrolyte species of varied hydrophobicity at the film’s surface. Fig. 2 summarizes the data on how the interior and the exterior of FP-containing films interact with water. Prior to the swelling experiments all the films were briefly dried in nitrogen gas flow at ambient temperature. Fig. 2A shows that, in good agreement with the film growth data, weakly bound PCPP/BPEI multilayers was able to uptake 45±4% of water, while FP60/BPEI multilayers accommodated a much smaller amount of water, which did not exceed 10% of their mass, when exposed to phosphate buffer, at pH 7.4. In comparison to FP-containing multilayers, weaker bound PCPP/BPEI multilayers have more polymer units not participating in the formation of polymer-polymer ionic pairs. The intrinsic hydrophilicity of the unbound polymer units, the osmotic pressure created by the counterions, and the electrostatic repulsion between excessive charges in the loops all contributed to an uptake of large amounts of water by the films. In contrast, as a polymer of a much lower polarity than PCPP, FP60 expelled water from the film, and created a low-dielectric-constant environment.44 Thus, strong electrostatic pairing occurred in all FP60-containing multilayers, resulting in the similarly low film swelling. Similarly, FP86-containg multilayers, which have an 11 ACS Paragon Plus Environment


increased of hydrophobic fluorinated groups, prevents water penetration and therefore suppresses swelling of FP86-containing multilayers (Fig. 2A).

Fig. 2. Swelling of LbL films formed by BPEI and FP60, FP86, or PCPP upon exposure to 0.01 M phosphate buffer at pH 7.4 (A); Contact angle variations during film buildup for various systems containing PPZs (B). Controlling surface hydrophobicity of non-covalent LbL assemblies might be challenging, as surface hydrophobic groups reorient into the film upon exposure to water.45 Fig. 2B shows, however, that large oscillations of the water contact angle with FP-containing multilayers occurred as a function of capping layer. The contact angle of water on a FP86-capped FP86/BPEI film was as high as 95°, and decreased to 55° when the top layer was changed to BPEI. Lowering the hydrophobic content (FP60/BPEI) within the system slightly decreased the maximum achieved contact angle (~80°). Similar behavior of alternating contact angles, with a smaller amplitude of oscillations, was observed with PPZ-containing systems with PLL as a partner (Fig. S2). In drastic contrast, PCPP-containing films exhibited a very small amplitude of the contact angle oscillations (~5°), and remained largely hydrophilic regardless of the top layer. Notably, oscillations in the contact angle in the FP60/BPEI and FP86/BPEI multilayers were not only high-amplitude, but also highly repeatable as a function of layer number (Fig. 2B). Until now, there have been only a few reports of the odd-even effect in surface adhesion, surface roughness, and swelling of polyelectrolyte multilayers.46-48 Interestingly, films of linearly growing, strongly bound polyallylamine hydrochloride and polystyrenesulfonate demonstrated significant oscillations in contact angle of a water droplet with layer number,49 while non-layer


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films formed by poly-L-lysine hydrobromide and poly-L-glutamic acid lacked oscillations in the contact angle amplitude.50 Compared to these earlier reports, iFP-containing multilayers showed a larger amplitude in the contact angle oscillations, and demonstrated “true” hydrophobic behavior with the contact angle above 90 oC. In order to understand the molecular origins of the robust linear growth of iFP-containing multilayers and the capping-layer-controlled localization of hydrophobic polyelectrolytes at the film surface, we performed neutron reflectometry (NR) studies of the iFP/polycation film structure. Moreover, keeping in mind the potential biomedical applications of iFP nanoassemblies, we have explored whether the structure of iFP-containing multilayers was sensitive to the presence of small ions in the environment at concentrations equal to or exceeding those regularly seen in biological fluids. In these experiments, assemblies of nonfluorinated polyphosphazene, PCPP, were used as a control.

Film internal structure: NR studies. NR is a versatile technique enabling studies of the internal structure of electrostatically31,40,51,52,53 or hydrogen-bonded54 LbL films. Here, we applied NR to characterize differences in the molecular binding and internal structure of films containing nonfluorinated and fluorinated PPZs. First, the effect of the type of PPZ was studied. The films were deposited using hydrogenated and deuterated QPC to provide contrast for NR measurements. The two-stack design was employed by our group previously and successfully used to quantify the dynamics of polyelectrolyte chains upon exposure to salt solutions.31 Fig. 3 shows the reflectivity data and fitted profiles for PCPPH/PCPPD and FP60H/FP60D multilayers (where PCPPH, PCPPD, FP60H and FP60D denote PCPP/QPC, PCPP/dQPC, FP60/QPC, and FP60/dQPC ten-bilayer stacks, respectively, see Experimental Section). The data for these films deposited from a lowsalt solution (0.01 M phosphate buffer) are represented by the black curves in Fig. 3. The reflectivity data were fitted using a model that consisted of a BPEI priming layer, covered with a hydrogenated stack and finally by a deuterated stack (Tables S1 and S2). NR reveals the scattering length density (SLD) of each stack in the model, its thickness, and the internal mixing between layers. The thickness of each stack within the two films were 14.4 and 15.8 nm for the PCPPH and PCPPD stacks, respectively, while thicknesses for FP60H and FP60D stacks were 10.9 and 8.3 nm, respectively. This result agrees well with the previously discussed ellipsometry results (Fig. 1D). Noticeably, the two systems show significant differences in the interfacial full width (σint) values, which characterize the degree of film intermixing. Thus, for FP60H/FP60D 13 ACS Paragon Plus Environment


Fig. 3. Reflectometry data (A, C) and scattering length density profiles (B, D) for FP60H/FP60D (top) and PCPPH/PCPPD (bottom) films deposited from 0.01 M phosphate buffer before and after 10-minute exposure to 0.4M NaCl solution in 0.01 M phosphate

buffer at pH 7.4. and PCPPH/PCPPD systems σint between hydrogenated and deuterated stacks increased from 3.8 to 10.6 nm, respectively, indicating strong intermixing for the PCPP-containing films. The large value of σint reflects the high mobility of PCPP and QPC chains during the LbL film buildup. It is interesting that the high mobility of polymer chains and non-linear growth of PCPP/QPC multilayers were observed here in spite of the spin-assisted deposition technique employed, which is known to enhance polyelectrolyte confinement and promote linear film growth.32 The persistence of the film layering in salt solution was then probed by exposing PCPPand iFP-containing films to 0.4 M NaCl solutions. Salt ions can disrupt polymer-polymer ionic pairing and promote polyelectrolyte diffusion.31 Fig. 3 shows SLD profiles for FP60H/FP60D and PCPPH/PCPPD (red and blue curves, respectively) before and after exposure to salt. Clearly, PCPP-containing multilayers were strongly affected by salt, showing an increase in the interfacial full width from 10.6 to 18.3 nm. Moreover, SLD of a hydrogenated block of this 14 ACS Paragon Plus Environment

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system increased from 1.57×10-6 Å-2 to 2.38×10-6 Å-2, indicating penetration of a significant amount of deuterated material (Tables S3 and S4). In contrast, the more layered FP60 remained relatively unaffected by the treatment with salt. Overall, NR studies have confirmed the superior layering and strong intermolecular binding within iFP-containing films. These molecular-level properties of the multilayers of fluorinated PPZs explain the observations of large-amplitude oscillations of the contact angle in Fig. 2. At the same time, high intermixing of more hydrophilic chains of non-fluorinated PPZs results in low values of the contact angles and the absence of the contact angle oscillations.



Fig. 4. Contact angle oscillations as a function of layer number for FP86/BPEI system on Ti foil (A). Polyester surgical felt (B) coated with 7 or 8 (terminated with PPZ or BPEI, respectively) layers of PPZ-containing films with a droplet of Rhodamine 6G solution (capping layer is denoted below each pad). Deposition of iFP LBL films on titanium and polyester substrates. Inspired by the possibility of controlling the wettability on planar surfaces via the deposition of LbL films of iFPs (Fig. 2B), we aimed to explore whether these films can be deposited on other, biomedically relevant materials. Two such materials are titanium and polyester felt, which are used commonly for orthopedic implants55 and surgical and medical device applications,56 respectively. Similarly to those deposited on silicon substrates, FP86-capped films coated on other substrates demonstrated high hydrophobicity (Fig. 4A). Furthermore, while the contact angle for the film on a titanium foil substrate was similar to that for a film on a silicon wafer (~90°, Fig. S3), the film on the porous surgical mesh had an enhanced value of 135°. This increase is explained by the porous structure (roughness) of the polyester surgical felt,57 that enhanced the contact angle for iFPcapped and decreased it for BPEI-capped coatings according to Wenzel’s equation.58 Importantly, similarly to FP86/BPEI multilayers on Si substrates, the contact angle oscillations with films deposited on Ti foil and polyester surgical felt were highly repeatable after three polyelectrolyte layers for a large number of deposition steps. An important feature of the PPZcontaining multilayers is its independence of surface wettability on the film thickness. FP86/BPEI systems deposited on titanium and surgical felt illustrated the robustness of PPZs LbL films and suggested that this system can be deposited on substrates of different chemistries. 16 ACS Paragon Plus Environment

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Fig. 4B illustrates the wettability properties of LbL-coated polyester surgical felt, and contrasts the use of fluorinated and non-fluorinated PPZs in these coatings. In these experiments, a small amount of Rhodamine 6G (~0.05 mg/mL) was added to water to enhance droplet visibility. The use of PCPP as a capping layer did not enhance the felt hydrophobicity, and the water droplet penetrated the mesh pad. In contrast, polyester surgical felt coated with iFP-containing film and terminated with either FP60 or FP86 were highly hydrophobic and retained a droplet of Rhodamine 6G at the surface of the sample. In all cases where the LbL coatings were terminated with polycation (BPEI), a droplet of Rhodamine 6G aqueous solution completely soaked the mesh pad. Thus, the developed coatings can be used to efficiently tune the wettability of surfaces. In addition, the inclusion of small molecules, illustrated here with a model Rhodamine 6G molecule, was enhanced within iFP/BPEI multilayers as compared to the LbL films of nonfluorinated PCPP (Fig. 4B), probably due to the additional contribution of hydrophobic interactions to small molecule absorption. Blood compatibility and selective protein adsorption. The major prerequisite for materials intended for life sciences applications is their adequate biocompatibility.59-61 Therefore, LbL films of iFPs as potential coatings for cardiovascular stents or catheters were evaluated using the widely accepted hemolysis test for evaluation of hemocompatibility of biomaterials.39, 59 It has been demonstrated that the superb hemocompatibility of clinically validated , PTFEP17, 20, 34 is correlated with its ability to selectively and irreversibly adsorb human serum albumin (HSA) from blood plasma.6 For that reason, the ability of fluorinated LbL nanofilms to bind HSA was also evaluated and compared to films of this non-ionic polyphosphazene, PTFEP, which was solution-cast from ethylacetate.

Fig. 5. Hemolysis of whole rabbit blood (A) and adsorption of HSA from human plasma (B) 17

on PPZs/BPEI multilayers and control solution-cast PTFEP coatings. ACS Paragon Plus Environment


Fig. 5A presents the results of a modified hemolysis test developed by the American Society for Testing and Materials conducted with dilute whole rabbit blood.39 In these experiments, the thicknesses of all coatings were matched (i.e. ~200 nm for solution-cast PTFEP coating, 20 bilayers of PPCP/BPEI, and 50 bilayers FP/BPEI films) and all LbL films contained PPZs as the top layer. As the content of fluorinated groups in iFP increased, i.e. PPZ polymer became more hydrophobic, hemolysis %, which represents an increase in the content of free hemoglobin in blood after incubation with the tested material, dramatically decreased. Remarkably, PPZ LbL multilayers assembled from aqueous solution using a PPZ with the highest content of fluorinated moieties (FP86) demonstrated comparable hemolysis content with a control, PTFEP coating. The results suggest that while the presence of hydrophilic carboxylic acid groups, which are required for polymer water-solubility and assembly of the coating, increase hemolysis, this effect is completely eliminated for polymers with a high content of fluorinated groups. In the FP86/BPEI multilayers, hemolysis-inducing carboxylic groups are strongly bound with amino groups of BPEI through hydrophobicity-enhanced ionic pairing, and buried within the FP86-capped film. Fig. 5B summarizes studies of selective adsorption of HSA from human plasma (reversible and irreversible) on the PPZ-containing multilayers and the control PTFEP coatings from human plasma. The extent of such adsorption has been previously related to passivation of surfaces against platelet adhesion.6 The percent of total HSA adsorption was dramatically higher for all PPZ-containing coatings as compared to the control PTFEP coating. Overall, the percent of irreversibly adsorbed HSA increased for more fluorinated iFPs, in good agreement with previous reports on the affinity of HSA to PTFEP investigated under similar conditions.6 Furthermore, irreversible HSA adsorption on LbL FP86/BPEI multilayers terminated with a layer of the highly fluorinated FP86 was higher than that observed for the control nonionic PTFEP coating. Therefore, ultrathin LbL films of fluorinated PPZs constructed in aqueous solutions can potentially achieve similar or even higher hemocompatibility compared to their solution-cast nonionic PTFEP counterpart.

Conclusion In this work, we report on biocompatible LbL films with controlled hydrophobicity and their interactions with biological milieu. The films can be electrostatically assembled using ionic 18 ACS Paragon Plus Environment

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fluorinated polyphosphazenes, iFPs, and a variety of polycations. Introduction of fluorinated groups into iFP played a critical role in achieving films with strong intermolecular binding and a high degree of layering, both of which are critical for controlling interfacial properties. The films built with more fluorinated PPZs were more stratified as shown by neutron reflectometry, and showed an extremely low uptake of water because of the hydrophobic environment within the films. In comparison, films formed by non-fluorinated ionic PPZs were intermixed, hydrophilic, and easily swollen by water. Most importantly, iFPs enabled construction of nanocotaings with controlled hydrophobicity and afforded a facile way of depositing conformal hydrophobic coating on several biomedically relevant planar and porous substrates through aqueous assembly. The hydrophobic LbL films capped with highly fluorinated iFPs demonstrated selective protein adsorption and exceptionally low hemolysis characteristics, suggesting levels of biocompatibility similar to or better than their non-ionic counterpart, PTFEP – a clinically validated material with superb biocompatibility. Considering the potential benefits of eliminating organic solvents from film processing, higher versatility, and the unprecedented level of controls afforded by the LbL technique, this approach can lead to a paradigm shift in the development of hemocompatible, fluorinated coatings.

Supporting Information Ellipsometric thicknesses for spin-deposited LbL films, contact angle data for PCPP/PLL and FP60/PLL multilayers, as well as SLD, block thicknesses and interfacial widths for FP60- and PCPP-containing LbL multilayers as determined by NR. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Authors *E-mails: [email protected]; [email protected]

Notes The authors declare no competing financial interest. For polyphosphazene-related inquiries, please contact A.K. Andrianov ([email protected]).

Acknowledgment 19 ACS Paragon Plus Environment


We acknowledge financial support from Texas A&M University/Association of Former Students Graduate Merit Fellowship. This work was supported in part by the National Science Foundation under Award DMR-1610725 (S.S.) and MPower Maryland (A.M. and A.A.), and intramural IBBR funding (A.M. and A.A.). Neutron measurements were performed at the Spallation Neutron Source at the Oak Ridge National Laboratory, managed by UT-Battelle, LLC, for the DOE under contract NO DE-AC05-00OR22725.

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For Table of Contents Only Biocompatible Nanocoatings of Fluorinated Polyphosphazenes through Aqueous Assembly Victor Selin1, Victoria Albright1, John F. Ankner2, Alexander Marin3, Alexander K. Andrianov3 and Svetlana A. Sukhishvili1* 1

Department of Materials Science & Engineering, Texas A&M University, College Station, Texas 77843, USA

2 3

Spallation Neutron Source, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA

Institute for Bioscience and Biotechnology Research, University of Maryland, Gaithersburg, MD, USA


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