Betulin-Constituted Multiblock Amphiphiles for Broad-Spectrum

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Betulin-Constituted Multiblock Amphiphiles for Broad-Spectrum Protein Resistance Ye Chen, Qilei Song, Junpeng Zhao, Xiangjun Gong, Helmut Schlaad, and Guangzhao Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16255 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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ACS Applied Materials & Interfaces

Betulin-Constituted Multiblock Amphiphiles for Broad-Spectrum Protein Resistance Ye Chen, † Qilei Song, † Junpeng Zhao,* † Xiangjun Gong, † Helmut Schlaad, ‡ and Guangzhao Zhang†

†

Faculty of Materials Science and Engineering, South China University of Technology, Guangzhou

510640, People’s Republic of China ‡

Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany

ABSTRACT. Multiblock-like amphiphilic polyurethanes constituted by poly(ethylene oxide) and biosourced betulin are designed for antifouling and synthesized by a convenient organocatalytic route comprising tandem chain-growth and step-growth polymerizations. The doping density of betulin (DB) in the polymer chain structure is readily varied by a mixed-initiator strategy. The spin-coated polymer films exhibit unique nanophase separation and protein resistance behaviors. Higher DB leads to enhanced surface hydrophobicity and, unexpectedly, improved protein resistance. It is found that the surface holds molecular-level heterogeneity when DB is substantially high due to restricted phase separation, therefore broad-spectrum protein resistance is achieved despite considerable surface hydrophobicity. As DB decreases, the distance between adjacent betulin units increases so that hydrophobic nanodomains are formed which provide enough landing areas for relatively small-sized proteins to adsorb on the surface.

KEYWORDS.

amphiphilic

surface,

antifouling,

multiblock

copolymer,

organocatalytic

polymerization, renewable resource

ACS Paragon Plus Environment

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INTRODUCTION Biofouling occurs widely and affects the performance of materials and devices used in contact with liquid biological systems. Typically, the sensitivity of sensor equipment used for tissue engineering, clinical diagnosis, and medical implants drops significantly in the presence of foulants.1, 2 Massivescale biofouling occurring on marine facilities, e.g. vessels and aquaculture systems, enhances corrosion and/or fuel consumption thus leads to increased operational and maintenance costs.3, 4 The process of protein deposition from the aqueous phase onto a solid surface is usually considered the initial step of the entire biofouling process. Therefore, great efforts have been made to develop surfaces which are resistant against protein adsorption.5-7 Hydrophilic poly(ethylene oxide) (PEO) has shown great utilities for fabrication of protein-resistant surfaces because of the effects of hydrated surface layer and/or steric repulsion it engenders as well as its non-toxicity and biocompatibility.8, 9 A major issue associated with such surfaces is that the hydration of PEO can lead to undesirable swelling and detachment of the coating from the substrate.10 Although covalently grafted PEO may refrain from such drawbacks, inconvenient preparation, high costs, poor mechanical properties, etc. may limit large-scale application. It is recognized that solely hydrophilic or hydrophobic surfaces might be incompetent for antifouling purposes due to the intrinsic amphiphilic nature of foulants.11, 12 For instance, bovine serum albumin (BSA) consisting of hydrophilic and hydrophobic patches is known to expose the matching patch towards the surface rendering adsorption.13 Ambiguous surfaces are thus currently in vogue, which have compositional heterogeneity, usually based on coexisting hydrophobic and hydrophilic components, to deter the settlement of a broader-spectrum of foulants.14, 15 Such surfaces have been mainly prepared by coating of polymer blends or amphiphilic block copolymers comprising immiscible components.16 The origin of fouling resistance of amphiphilic ambiguous surface has been


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revealed as weakened thermodynamic interactions with protein and minimized protein conformational re-organization.12 A major concern is that self-assembly (phase separation) driven by the incompatibility of components of different natures may actually induce protein adsorption especially when the hydrophobic domains exceeds the size needed for the settlement of proteins.17 Therefore, surfaces with nano-scaled morphological heterogeneities have been designed and fabricated to create a dimensional mismatch between the nanodomains and anchoring sites for proteins.18, 19 In particular, molecular-level heterogeneity appears even more competent for broad-spectrum protein resistance.20, 21

, Ambiguous surfaces fabricated by amphiphilic di- or triblock copolymers or polymer blends

usually display complex surface morphologies at nano-to-micro length scales.22, 23 We have envisioned that (AB)n type amphiphilic multiblock copolymers would favor low-scale or even molecular-level heterogeneity due to poor mobility and restricted aggregation of the structural segments.24, 25 Previously, a bioconjugate polymer comprising a rigid-hydrophobic betulin entity in the center and soft-hydrophilic PEO chains on both sides (BEO), was synthesized by metal-free anionic ringopening polymerization (ROP).26 Interestingly, such an amphiphilic triblock-like structure, when dissolved in water, stays mostly as single molecules rather than form stable nano-sized aggregates despite substantially high hydrophobic content, which is most probably related to the viscoelastic effect (longer relaxation time than interaction time) derived from the peculiar molecular geometry and the rigidity of betulin units.27, 28 By reacting with an diacid chloride, BEO was turned into a segmented polyester and lost the solubility in water.29 It was anticipated that such a betulin-constituted multiblock-like structure might lack the ability to phase-separate and form hydrophobic nanodomains in its bulk. Together with the amphiphilicity and water-insolubility, this character would then lead to a competent protein-resistant coating material. In this study, we have altered the ester linkages to


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carbamates which would afford adequate chemical stability for underwater applications as well as good adhesion to substrates.30

EXPERIMENTAL SECTION Materials Tetrahydrofuran (THF) and toluene (Guangzhou Chemical Reagent, 99%) were dried successively by calcium hydride (CaH2) and n-butyllithium (n-BuLi) before distilled. Dichloromethane (DCM; Guangzhou Chemical Reagent, 99%) was dried by CaH2 and distilled. Betulin, 1,4-benzenedimethanol (BDM), 1,4-butanediol (BDO), isophorone diisocyanate (IPDI), and hexamethylene diisocyanate (HDI) were purchased from Aladdin. Betulin and BDM were dried in vacuum at 60 °C overnight. BDO were dried over CaH2 and distilled under vacuum. IPDI and HDI were stored in an inert atmosphere and used without further purification. Diphenyl phosphate (DPP; 99%), t-BuP4 (0.8 M in n-hexane), ethylene oxide (EO; 99.5%), and 1,2-butylene oxide (BO; 99%) were purchased from Aldrich. DPP was dried by azeotropic distillation of toluene, then dissolved in purified toluene into a 0.5 M solution. t-BuP4 was used as received. EO was condensed in a Schlenk flask docked on the vacuum line and dried by stirring with sodium hydride (NaH) in an ice-water bath for 4 h. BO was stirred with NaH and then cryo-distilled on the vacuum line. Instrumentation NMR spectra were recorded at 25 °C on a Bruker AV400 NMR spectrometer using deuterated chloroform (CDCl3) as the solvent and tetramethylsilane as the internal standard. For some of the polymers, 1H NMR spectra were used to calculate number-average molar mass (Mn,NMR) by comparing the integrals of signals from end groups and polymer main bodies. Size exclusion chromatography (SEC) coupled with refractive index (RI) and UV detectors was conducted in N,N-dimethylformamide (DMF) with LiBr (0.05 M) at 50 °C and a flow rate of 1.0 mL min-1 using three successively


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connected Styragel columns (HR2, HR4, HR6). For some of the polymers which are not well soluble in DMF, SEC was conducted in THF at 35 °C using two identical PLgel MIXED-C columns at the same flow rate. Calibration was done with a series of narrowly dispersed polystyrene (PS) or PEO standards to obtain apparent number-average molar mass (Mn,SEC) and molar mass distribution (ĐM) of the polymers. Differential scanning calorimetry (DSC) measurements were performed on a NETZSCH DSC204F1 system in a nitrogen flow. The polymer was quickly heated to 150 °C, kept at this temperature for 4 min to remove thermal history, cooled to −80 °C at a cooling rate of 10 °C min-1, and then heated again to 150 °C at a heating rate of 10 °C min-1. Thermogravimetric analysis (TGA) was conducted on a NETZSCH STA449C thermal analyzer under a nitrogen atmosphere at a heating rate of 10 °C min-1 in the temperature range of 25-600 °C. The polyurethane (PU) films were prepared by spin-coating of toluene solution (5.0 mg mL-1) on a spin-coater (CHEMAT, KW-4) at 600 rpm for 9 s and then 2000 rpm for 1 min in air. Contact angle (CA) measurements were conducted on an OCA40 instrument (DataPhysics) by depositing 3 µL water droplets on a PU surface using the sessile method at 25 °C. The average of results from three measurements was used as the final CA value. The adsorption of proteins was monitored with quartz crystal microbalance with dissipation (QCM-D), which together with AT-cut quartz crystal with a fundamental resonance frequency of 5 MHz was from Q-sense AB. Briefly, the mass gain/loss of a thin layer on a quartz crystal is manifested by the decrease/increase in the resonance frequency of the crystal, whereas the dissipation factor is related to the viscoelastic properties of the additional layer. In the present study, phosphate buffer saline (PBS) was used as the reference. The spin-coated polymer films were immersed in PBS buffer for at least 12 h before adsorption study. Lysozyme (Lys.), BSA, and fibrinogen (Fib.) with different sizes and isoelectric points were dissolved in PBS (1.0 mg mL-1) and stood for 2 h before use. The protein solution was delivered to the surface at a flow rate of 150 µL min-1. The changes in frequency (∆ƒ) and


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dissipation (∆D) gave information about the protein adsorption. All the experiments were performed at 25 °C, and the data presented were from the third overtone. The PU films formed on gold-coated quartz crystal surfaces were imaged on an XE-100 Atomic Force Microscope (AFM, Park Systems) in air or PBS. The surface morphology was obtained in tapping mode with an NCHR cantilever (spring constant 42 N m-1). Polymer Synthesis Two-Pot Route to Betulin-Doped Poly(ethylene oxide)-Polyurethane (BEO-PU) Typically for BEO2K-PU (Table 1, S1). Betulin (1.0 g, 2.26 mmol) was loaded in the reaction flask, purified by cryo-evaporation of THF, and dried in vacuum for 1 h at 60 °C. THF (50 mL) was then condensed into the flask to completely dissolve betulin. t-BuP4 solution (0.28 mL, 0.22 mmol, 0.05 equiv. with respect to hydroxyl) was added in an argon flow. The flask was cooled at −20 °C and purified EO (5.2 mL, 104.0 mmol) was introduced by cryocondensation. Then the reaction mixture was slowly heated to 45 °C and stirred for 48 h before quenched with acetic acid. After evaporation of THF, the crude product was dissolved in water and stirred with ion-exchange resin (Dowex@50WX4100) for 15 min to remove the phosphazenium salt. Then the resin was filtered off and the final product (BEO2K) was isolated by freeze-drying. Mn,SEC(DMF, PS standards)=7.9 kg mol-1, ÐM=1.08; Mn,SEC(THF, PEO standards)=2.1 kg mol-1, ÐM=1.30; 1H NMR (400 MHz, CDCl3), 4.62-4.48 (betulin, =CH2), 3.80-3.35 (PEO, −OCH2CH2O−), 3.10-3.04 (betulin, −CH2OCH2CH2O−), 2.73-2.65 (betulin, −CHOCH2CH2O−); Mn,NMR=2.3 kg mol-1. The isolated BEO2K (0.50 g, 0.22 mmol) was dissolved in toluene and loaded in a reaction flask. After cryo-evaporation of toluene and drying in vacuum at 60 °C for 2 h, BEO2K was dissolved in purified toluene (20 mL) cryo-condensed into the flask. Then IPDI (0.046 mL, 0.22 mmol) and DPP solution (0.044 mL, 0.022 mmol) were added. The step growth polymerization (SGP) was allowed to proceed


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at 60 °C for 3 days. Then the reaction was quenched by addition of triethylamine and the product was precipitated in a mixture of methanol and diethyl ether (1/1, v/v). Mn,SEC(DMF, PS standards)=50.4 kg mol-1, ÐM=1.56; 1H NMR (400 MHz, CDCl3), δ/ppm=4.62-4.48 (betulin, =CH2), 4.30-4.05 (PEO, −CH2CH2OCONH−), 3.80-3.35 (PEO, −OCH2− and IPDI, −CHNHCOO−), 3.10-3.04 (betulin, −CH2OCH2CH2O−), 2.87-2.80 (IPDI, −CH2NHCOO−), 2.73-2.65 (betulin, −CHOCH2CH2O−). Betulin-Doped Poly(ethylene oxide)-Polyurethane with Chain Extender (BEO-PUex) Typically for BEO2K-PUex (Table S1). After purification and dissolution of BEO2K (0.50 g, 0.22 mmol) in toluene (20 mL) in the reaction flask, IPDI (0.092 mL, 0.44 mmol) and DPP solution (0.088 mL, 0.044 mmol) were added. The flask was then heated at 60 °C for 24 h, then BDO (0.019 mL, 0.22 mmol) was added to act as a chain extender. After heating at 60 °C for another 48 h, the reaction was finally quenched by addition of triethylamine and the product was precipitated in a mixture of methanol and diethyl ether (1/1, v/v). Mn,SEC(DMF, PS standards)=71.9 kg mol-1, ÐM=1.93; 1H NMR (400 MHz, CDCl3), δ/ppm=4.60-4.47 (betulin, =CH2), 4.30-4.08 (PEO, −CH2CH2OCONH−), 4.073.95 (BDO, −OCH2CH2CH2CH2OCONH−), 3.80-3.35 (PEO, −OCH2− and IPDI, −CHNHCOO−), 3.10-3.04 (betulin, −CH2OCH2CH2O−), 2.87-2.80 (IPDI, −CH2NHCOOCH2CH2O−), 2.75-2.60 (betulin, −CHOCH2CH2O− and IPDI, −CH2NHCOO(CH2)4O−). One-Pot Route to BEO-PU Typically for B30EO2.5K-PU (Table 1). BDM (0.193 g, 1.40 mmol) and betulin (0.266 g, 0.60 mmol) was added in a Schlenk flask, purified by azeotropic distillation of THF and dried in vacuum at 60 °C for 1 h. Then purified toluene (20 mL) was condensed in the flask, followed by addition of t-BuP4 solution (0.28 mL, 0.22 mmol, 0.05 equiv. with respect to hydroxyl) in an argon flow. The flask was cooled at −20 °C and purified EO (5.7 mL, 5.0 g, 113.6 mmol) was introduced by cryocondensation. Temperature was then raised to 45 °C and the reaction mixture was magnetically stirred. The


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suspended initiator powders dissolved in 30 min indicating the polymerization of EO occurred therefrom. After 48 h, a small aliquot was withdrawn in an argon flow and quenched with acetic acid for SEC analysis. Then DPP solution (2.0 mL, 1.0 mmol) was added to the reaction mixture to effect catalyst switch. After stirring for 30 min, IPDI (0.42 mL, 1.98 mmol) was added and temperature was raised to 60 °C to start the SGP which was quenched after 3 days by addition of triethylamine followed by precipitation of the product in a mixture of methanol and diethyl ether (1/1, v/v). B30EO2.5K precursor: Mn,SEC(DMF, PS standards)=8.6 kg mol-1, ÐM=1.06; Mn,SEC(THF, PEO standards)=2.2 kg mol-1, ÐM=1.06. B30EO2.5K-PU: Mn,SEC(DMF, PS standards)=88.2 kg mol-1, ÐM=1.67; 1H NMR (400 MHz, CDCl3), δ/ppm=7.25-7.19 (aromatic protons), 4.60-4.47 (betulin, =CH2), 4.55-4.45 (BDM, −OCH2C6H4CH2O−), 4.30-4.08 (PEO, −CH2CH2OCONH−), 3.80-3.35 (PEO, −OCH2− and IPDI, −CHNHCOO−), 3.10-3.04 (betulin, −CH2OCH2CH2O−), 2.87-2.80 (IPDI, −CH2NHCOO−), 2.75-2.60 (betulin, −CHOCH2CH2O−). One-Pot Route to Betulin-Doped Poly(1,2-butylene oxide-co-ethylene oxide)-Polyurethane The synthesis of BBO15EO85-PU (Table S1) followed a similar procedure as described above for B30EO2.5K-PU, using betulin (1.0 g, 2.26 mmol), toluene (20 mL), t-BuP4 solution (0.28 mL, 0.22 mmol), EO (5.0 mL, 100.0 mmol), BO (1.5 mL, 17.3 mmol), DPP solution (2.26 mL, 1.13 mmol), and IPDI (0.48 mL, 2.27 mmol), except that EO and BO were purified separately, mixed in one flask and cryo-distilled together into the reaction flask. BBO15EO85 precursor: Mn,SEC(THF, PEO standards)=1.8 kg mol-1, ÐM=1.04. BBO15EO85-PU: Mn,SEC (THF, PEO standards)=18.8 kg mol-1, ÐM=1.56; 1H NMR (400 MHz, CDCl3), δ/ppm=4.87-4.73 (BO, −OCH2CH(CH2CH3)OCONH−), 4.68-4.53 (betulin, =CH2), 4.25-4.22 (EO, −OCH2CH2OCONH−), 3.78-3.35 (PEO, −OCH2− and IPDI, −CHNHCOO−), 3.10-3.04 (betulin, −CH2OCH2CH2O−), 2.87-2.80 (IPDI, −CH2NHCOO−), 2.75-2.60 (betulin, −CHOCH2CH2O−).


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One-Pot Route to Betulin-Doped Poly(1,2-butylene oxide)-Polyurethane The synthesis of BBO-PU (Table S1) followed a similar procedure as described above for BBO15EO85-PU, except that only BO was used as the epoxide monomer, using betulin (1.0 g, 2.26 mmol), toluene (10 mL), t-BuP4 solution (0.28 mL, 0.22 mmol), BO (6.80 mL, 78.3 mmol), DPP solution (2.26 mL, 1.13 mmol) and IPDI (0.48 mL, 2.29 mmol). BBO precursor: Mn,SEC(THF, PEO standards)=1.9 kg mol-1, ÐM=1.10. BBO-PU: Mn,SEC(THF, PEO standards)=19.8 kg mol-1, ÐM=1.97; 1

H NMR (400 MHz, CDCl3), δ/ppm=4.84-4.70 (PBO, −OCH2CH(CH2CH3)OCONH−), 4.69-4.53

(betulin, =CH2), 3.85-3.25 (PBO, −OCH2CH(CH2CH3)O− and IPDI, −CHNHCOO−), 3.08-3.04 (betulin, −CH2OCH2CH(CH2CH3)O−), 2.75-2.68 (betulin, −CHOCH2CH(CH2CH3)O−), 2.63-2.61 (IPDI, −CH2NHCOO−). Betulin-Polyurethane Procedure for B-PU (Table 1). Betulin (1.00 g, 2.26 mmol) was added in a reaction flask, purified by azeotropic distillation of THF and dried at 60 °C under vacuum for 1 h. Dried DCM (20 mL) was then cryo-condensed in the flask, followed by addition of HDI (0.36 mL, 2.26 mmol) and DPP solution (1.8 mL, 0.9 mmol). The flask was heated at 40 °C and soon after which the suspended betulin powder dissolved. The reaction was quenched after 3 days and the product was precipitated in methanol. Mn,SEC(THF, PEO standards)=16.9 kg mol-1, ÐM=1.34; 1H NMR (400 MHz, CDCl3), δ/ppm=4.60-4.47 (betulin, =CH2), 4.39-4.30 and 3.9-3.78 (betulin, −CH2OCONH−), 4.30-4.21 (betulin, −CHOCONH−) and 3.23-3.08 (HDI, −CH2CH2CH2NHCOO−).

RESULTS AND DISCUSSION A conventional two-pot multistep synthetic route was first used to obtain the designed betulin-doped amphiphilic polyurethane with PEO and betulin units occurring alternately and repeatedly along the main chain (BEO-PU; Scheme 1a and S1a). Namely, anionic ROP of EO with betulin as a difunctional


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inititaor is performed first followed by isolation and purification of the betulin-PEO precursor which is then subjected to SGP with a diisocyanate under a different catalytic condition. In addition to a tedious operation procedure, this method may also pose difficulties in ensuring equal numbers of hydroxyl and isocyanate groups which is crucial for the SGP step. The recent advancement of organocatalytic polymerization has provided new opportunities to fulfil such synthetic tasks in more facile and exquisite manners.31, 32 Herein, a previously developed catalyst switch strategy is well applied on the BEO-PU structure through one-pot sequentially performed base-catalyzed ROP and acid-catalyzed SGP (Scheme 1b).33 Thin films with reproducible surface morphology can be easily prepared by spincoating of the BEO-PU products. Table 1 and S1 list the characteristics of PU samples synthesized via either two-pot or one-pot route and the properties of the PU films.

Scheme 1. Schematic illustration for the synthesis of betulin-doped poly(ethylene oxide)-polyurethane by (a) the two-pot route (via isolation of BEO precursor) and (b) the one-pot route (sequentially performed ROP and SGP through the base-to-acid catalyst switch).


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Table 1. Molecular characteristics and properties of the betulin-doped polyurethanes.a entry

Mn,SECd -1

(kg mol )

ÐMd

WEOe

CA

(%)

(°)

RLys f

RBSAf

RFib.f

B-PU

16.9

1.34

0

87

0

0

0

BEO1K-PUb

69.1

1.65

60

86

0.93

>0.99

>0.99

BEO2K-PUb

50.4

1.56

73

78

>0.99

>0.99

>0.99

B10EO1.5K-PUc

79.0

1.43

75

31

0.46

0.89

0.97

B10EO2.5K-PUc

55.2

1.67

85

23

0.69

0.93

>0.99

B30EO2.5K-PUc

88.2

1.67

83

37

0.65

>0.99

0.98

B100EO2.5K-PUc

83.3

1.62

77

78

>0.99

>0.99

>0.99

a

Synthesized by one-pot sequentially performed organocatalytic ROP of EO (45 °C, 48 h) and SGP of the

functional PEO diol with IPDI (60 °C, 72 h) in toluene; bBEO1K-PU denotes that the theoretical numberaverage molecular weight of PEO in each BEO repeat unit (two PEO chains combined) is ca. 1 kg mol-1. c

B10EO1.5K-PU denotes that 10 mol% of the BEO precursors were initiated by betulin (and the other 90

mol% by BDM; DB=0.1). dApparent number-average molar mass and molar mass distribution determined by SEC (in THF at 35 °C for B-PU and in DMF at 50 °C for others using polystyrene standards). eWeight percentage of PEO calculated from 1H NMR spectra of the isolated products. fEffectiveness of protein resistance quantified as 1-∆ƒBEO-PU/∆ƒB-PU, with ∆ƒ values obtained in QCM experiments after rinsing.

For comparison, PUs without betulin doping (EO-PU) are synthesized from commercial PEO diols, and a solely hydrophobic PU is synthesized by reacting betulin with a diisocyanate (B-PU; Scheme S1b). Due to the relatively poor reactivity of betulin secondary hydroxyl,34 less sterically hindered hexamethylene diisocyanate is used for B-PU while isophorone diisocyanate is used for the others. The increased molar masses (Table S1) shown by SEC and the presence of all expected signals in 1H NMR spectra of the final products (Figure S1-S3) demonstrate the success of turning (macro)diol precursors into PUs. As shown in DSC traces (Figure S4), bulky and rigid betulin units in the PU structure hinder the crystallization of PEO (EO2K-PUex vs. BEO2K-PUex) and elevates the glass transition temperature (Tg; EO1K-PUex vs. BEO1K-PUex). Tg is also higher when BDO is used as a chain


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extender as it increases the density of hydrogen-bond-forming carbamate groups in the polymer structure (BEO1K-PUex vs. BEO1K-PU). However, this effect is much less profound when PEO chains are longer (BEO2K-PUex vs. BEO2K-PU). Thermogravimetric analysis shows that thermostatbility of PEO-based PUs is enhanced by incorporation of betulin units (also see Table S1). Water contact angles of the spin-coated BEO-PU films are much higher than those of EO-PUs and close to that of B-PU (Table S1), indicating that the surfaces of BEO-PU films are substantially hydrophobic in spite of high weight percentages of PEO (WEO>60%, Table 1). Increasing the molar mass of PEO from 1 kg mol-1 to 2 kg mol-1 (two segments combined) does not induce a significant decrease in CA (BEO2K-PU vs. BEO1K-PU; BEO2K-PUex vs. BEO1K-PUex). QCM-D is utilized to monitor the surface adsorption of Lys., BSA, and Fib., which are used as typical proteins with different molecular sizes and isoelectric points (Table S2) to estimate the protein resistance performance of antifouling surfaces.35 Evident drops of ∆ƒ and increases of ∆D (for BSA and Fib.) are observed when B-PU films are treated with protein solutions and then rinsed with PBS (Figure 1), indicating extensive adsorption and robust adhesion of all three proteins most probably through hydrophobic interaction with betulin units.36 On the contrary, BEO-PU films treated with all three proteins show almost zero ∆ƒ and ∆D changes indicating highly effective and broad-spectrum protein resistance of such surfaces, which, at this point, appears quite surprising in view of the considerable betulin content and surface hydrophobicity as reflected by CA.


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Figure 1. Time dependence of frequency shift (∆ƒ) and dissipation shift (∆D) from QCM-D studies for protein adsorption in PBS at 25 °C on amphiphilic films of BEO-PUs synthesized by the two-pot route, with B-PU film used as a hydrophobic reference. We have then assumed that the such properties of the BEO-PU films are associated with their surface morphology. Namely, molecular-level heterogeneity is likely to be maintained due to the unique macromolecular structure in which every two adjacent betulin units are separated by two short PEO chains hence phase separation and formation of hydrophobic domains are sterically restrained. To verify this assumption and demonstrate the effect of surface morphologies of BEO-PU films on protein resistance, comparative study needs to be carried out using BEO-PUs with different distances between adjacent betulin units, described herein as betulin doping density (DB). To obtain different DBs, variation of the average length of PEO segments by controlling the feed ratio of betulin and EO in the ROP step appears to be a straightforward means (Scheme 1a). But in this way the amount of diisocyanate used and carbamate linkage generated would have to be altered. Moreover, when a low DB was targeted, molar mass of PEO would likely be too high to ensure sufficient reactivity of the hydroxyls towards the SGP reaction, and long PEO chains would have high crystallinity which is unfavorable for surface coating materials. Therefore, a mixed initiator strategy is developed and used herein. Namely, betulin and a regular small-molecule diol, i.e. BDM, are mixed in


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a known molar ratio and used together to initiate the ROP of EO, so that the final PU are constituted by two randomly distributed PEO segments with betulin- and BDM-derived centres. It is reasonable to assume that the terminal hydroxyls of the polyether precursors are equally reactive and the SGP occurs in a random manner regardless of the different bulkiness of the central groups because of the existence of PEO spacers. In this way, DB=[betulin]0/([betulin]0+[BDM]0) is readily varied and long PEO chains are avoided even for low DB. For this series of BEO-PUs (Table 1), the one-pot route via a base-toacid catalyst switch process is used which allows convenient synthesis of the seemingly complicated macromolecule (Scheme 1b). The successful synthesis of the designed macromolecular structures is well demonstrated by SEC and 1H NMR analysis (Table 1, Figure 2, S5, and S6). The structure of betulin-doped PU can be further enriched by replacing EO with other epoxides, e.g. BO, and mixed monomers, e.g. BO and EO (Scheme 1b, Table S1, Figure S7 and S8).

Figure 2. 1H NMR spectrum of a representative betulin-doped polyurethane synthesized via the onepot route (B30EO2.5K-PU, Table 1). As is shown in Table 1, CAs of B10EO-PU2.5K and B10EO-PU1.5K films (DB=0.1) are 23° and 31°, respectively, whereas B100EO-PU2.5K (DB=1.0) surface appears to be much more hydrophobic


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(CA=78°; the same as BEO2K-PUs discussed above) though WEO is not significantly different. CA of B30EO-PU2.5K film is in between. This result indicates that the hydrophilic-hydrophobic content may not be the only factor that controls overall surface hydrophilicity. Surface morphologies of BEO-PU films with different DB are then investigated by atomic force microscope (Figure 3, S9, and S10). Numerous bright and nano-sized domains are clearly visualized in the phase and topology images of BEO-PUs with DB=0.1 both in air and PBS, while B100EO-PU2.5K with DB=1.0 presents smooth surfaces despite a higher betulin weight percentage. B30EO-PU2.5K seems to show an intermediate phase separation behaviour.

Figure 3. Structural illustration of BEO-PUs with different DBs (upper); AFM phase images (lower) and water contact angle (insets) of spin-coated films in air. None of the BEO-PUs is soluble or remarkably swollen in water or PBS regardless of DB and PEO lenghths (B100EO-PU2.5K practically does not swell at all). AFM results indicate that molecularlevel heterogeneity is maintained in BEO-PU film when DB is substantially high, and nanophase separation (aggregation of betulin units), driven by the incompatibility of the two major components


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(Figure S11), is facilitated as DB decreases probably because of the extended average distance between two adjacent betulin units (Figure 3). The phase-separated surface presents a low CA because the continuous phase consists mainly of hydrophilic PEO and covers >97% of the total area (in dry state; calculated by ImageJ),37 while the large amount and molecular-level dispersion of betulin units in the smooth surface leads to weakened overall interaction of PEO with water molecules and a consequently higher CA. The roles that BDM and carbamate moieties are playing are not taken into consideration in the present discussion due to much lower mass contributions.

Figure 4. Time dependence of frequency shift (∆ƒ) and dissipation shift (∆D) from QCM-D studies for protein adsorption in PBS at 25 °C on amphiphilic films of BEO-PUs with different PEO lengths and DBs synthesized via the one-pot route (Table 1). B-PU film is used as a hydrophobic reference. The next question hence arises whether the different phase behaviour would have an impact on protein resistance. Figure 4 presents the QCM-D results for protein adsorption on BEO-PU films with different DBs. Regardless of a higher hydrophobic content and a higher CA, B100EO2.5K-PU (DB=1.0) shows practically zero frequency shifts for Lys., BSA, and Fib. after rinsing (also see Table 1), indicating complete resistance towards these proteins. On the phase-separated surfaces, the largest protein (Fib.) is still fully resisted, whereas the adsorption of smaller ones (Lys. and BSA) is clearly indicated, though not as profound as the adsorption on the solely hydrophobic reference (B-PU film).


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Note that for the smallest Lys., reduction of adsorption relative to B-PU film (RLys.) reaches 89% on such surfaces (Table 1). Though the recognition between protein and surfaces occurs at much smaller length scales, substantially large “landing areas”, which are usually nano-to-micro-scaled hydrophobic domains, are needed for proteins to adsorb firmly on the surface.20 Therefore, the surface of B100EO2.5K-PU film, holding molecular-level heterogeneity, completely resists even the smallest protein investigated (Lys.). The phase-separated surfaces (low DB) provide such landing areas for Lys. and BSA, which, however, are not large enough for Fib.38 To better understand the roles that the two factors, i.e. amphiphilicity and molecular-level heterogeneity, play for protein resistance, hydrophilic PEO segments are replaced by hydrophobic poly(1,2-butylene oxide) or partially hydrophobic poly(1,2-butylene oxide-co-ethylene oxide) (BBOPU and BBO15EO85-PU in Table S1; DB=1.0) with similar segmental molar masses as B100EO2.5K-PU. Nanophase separation is not observed by AFM for the films of such polymers (Figure S12). QCM-D study shows that for all the three proteins, adsorption that occurs on BBO-PU films is profound and comparable to B-PU films (Figure S13). On the other hand, protein-resistant capabilities are almost fully preserved when the polyether segments consist only 15 mol% of BO-derived monomeric units. Such results have clearly demonstrated that both amphiphilicity and maintained molecular-level heterogeneity are essential factors for ensuring broad-spectrum protein resistance, and the unique multiblock-like PEO-PU structure with densely doped betulin units is therefore successfully designed and synthesized to fulfill such requirements.

CONCLUSIONS In summary, a one-pot organocatalytic route has been successfully used for facile construction of novel betulin-doped amphiphilic and multiblock-like polyurethanes. The structural characteristics (DB,


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length and composition of polyether segments) can be readily controlled for modulating the properties of the spin-coated films. When DB is substantially high, molecular-level heterogeneity is maintained in the film so that broad-spectrum protein resistance is achieved despite considerable overall hydrophobicity. As DB decreases, the extended distance between adjacent betulin units facilitates phase separation so that hydrophobic nanodomains form which act as landing areas for relatively small-sized proteins. The convenient synthesis, partially bio-renewable feature, variable nanophase separation and protein resistance behaviour are believed to make such polymers competent for antifouling or other applications that require tunable protein-surface interactions. Further studies are being carried out towards more quantitative understanding of the relationships of macromolecular structure, thin film composition and preparation method, phase separation, and interaction with biological entities (e.g. proteins and bacteria).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional reaction scheme, polymer characterization data, AFM images, and QCM-D traces for protein adsorption studies.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ACKNOWLEDGMENTS The financial support of National Natural Science Foundation of China (21674038, 21734004) and Fundamental Research Funds for Central Universities (2017ZD072) is acknowledged.


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For Table of Contents Use Only


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TOC graphic 79x42mm (300 x 300 DPI)



Scheme 1 160x130mm (300 x 300 DPI)


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Figure 1 160x81mm (300 x 300 DPI)



Figure 2 80x57mm (300 x 300 DPI)


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Figure 3 160x115mm (300 x 300 DPI)



Figure 4 160x81mm (300 x 300 DPI)


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Betulin-Constituted Multiblock Amphiphiles for Broad-Spectrum

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