Self-Healable Antifouling Zwitterionic Hydrogel Based on Synergistic

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Applications of Polymer, Composite, and Coating Materials

Self-healable Antifouling Zwitterionic Hydrogel Based On Synergistic Phototriggered Dynamic Disulfide Metathesis Reaction and Ionic Interaction Sovan Lal Banerjee, Koushik Bhattacharya, Sarthik Samanta, and Nikhil Kumar Singha ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10446 • Publication Date (Web): 18 Jul 2018 Downloaded from http://pubs.acs.org on July 23, 2018

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ACS Applied Materials & Interfaces

A Self-healable Antifouling Zwitterionic Hydrogel Based On Synergistic Photo-triggered Dynamic Disulfide Metathesis Reaction andIonic Interaction SovanLal Banerjee, Koushik Bhattacharya, SarthikSamanta, Nikhil K Singha* Rubber Technology Centre, Indian Institute of Technology, Kharagpur, India.

Abstract: A self-healable antifouling hydrogel based on zwitterionic block copolymer was prepared via Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization and Diels-Alder “click” chemistry. The hydrogel consists of a core-crosslinked zwitterionic block copolymer having poly(furfuryl methacrylate) (PFMA) as a core and poly(dimethyl-[3-(2-methylacryloylamino)-propyl]-(3-sulfopropyl)ammonium) (poly(sulfobetaine), PSB) as a shell. The core was crosslinked with dithiobismaleimidoethane (DTME). The block copolymers (BCPs) were characterized by DLS, FESEM, HRTEM, AFM, DSC, WCA and SAXS analyses. This zwitterionic hydrogel showed self-healing activity via combined effect of photo-triggered dynamic disulfide metathesis reaction and zwitterionic interaction which was monitored by optical microscopy and AFM depth profilometry. Mechanical properties of the hydrogel before and after the self-healing were studied using depth sensing nano-indentation (DSI) method. It was observed that the prepared zwitterionic hydrogel could reduce the formation of biofilm which was established by studying the Bovine Serum Albumin (BSA-model protein) adsorption over the coating. This multifunctional hydrogel can pave a new direction in the antifouling selfhealable gel coating applications. Keywords: Hydrogel, RAFTpolymerization, Diels-Alder, Block copolymer, zwitterion, Selfhealing, antifouling.

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Introduction: Biofouling or biocontamination is a major problem in many sectors including textile1, 2, food packaging3, 4, biosensors5, water purification system6, marine industry7, 8, medical goods such as catheters9, contact lenses10, 11, surgical equipment12 etc. Biofouling stands for the non-specific adsorption of the protein and microbes over the surface which lead to the deterioration of the efficiency of products, harmful side effects and endowa friendly surface to the microbial growth. Microbial growth over the surface or biofilm formation can cause severe health hazards leading to costly treatments. Biofouling is also a significant concern for the marine industries as the protein deposition over the surface can result inincreased rate of deposition of the other aquatic species like algae, fungus etc.7 which affect the coating and can generate various surface defects. Now-a-days there is a great demand for antifouling smart polymer coatings that can effectively prevent adhesion of the protein over the surface. Poly(ethylene glycol) (PEG) based polymers are extensively used as an antifouling coating material due to their high hydrophilicity as well as non-toxic nature13, 14. Along with PEG, some of the polyhydroxylated polymers are also able to show antifouling activity via forming interfacial water layer15, 16. Prime17, 18 and Whitesides18, 19 reported an oligo(ethylene oxide) based self-assembled monolayers (SAMs) that can efficiently reduce the protein adhesion. Grunzeetet.al.19 reported that the conformation of the oligo(ethylene glycol) also plays a vital role over the protein adhesion. Although PEG-based smart polymer coating material has many advantages, but it has some drawbacks like auto-oxidation of the hydroxyl groups to form the aldehyde and ether which lead to the loss of anti-fouling activity20. So a new class of antifouling agent is required which can effectively resist the protein adhesion as well as chemical deterioration. Jiang et al. reported that poly(zwitterionic) materials could show an ultra low-fouling. It was reported that the Zwitterionic sulfobetaines could show a very low level of protein adsorption (< 0.3 ng.cm-2) and also show excellent hydrophilicity, saltresistance property and biocompatibility21-24. Along with the antifouling property, self-healing is a significant point of concern in the coating industry. Generation of the micro-cracks can result in a catastrophic failure of the product leading to the decrease in the shelf-life of the product. So, healing of the generated micro-cracks is necessary for the long-term stability of the product. Self-healing can be of two types- i) intrinsic self-healing which doesn’t require any external stimuli for healing and ii) induced self-


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healing which requires external stimuli like heat25, pH26, 27etc. Self-healing coating is very much important for the coating in less accessible and integrated areas. The first reported self-healing material was based on the nano/microcapsule system bearing the healing monomer and catalyst. On crack generation, these capsules burst after that the catalyst and the monomer are released, and then the healing takes place because of the chemical reaction28. In this case, the major problem is the irreversibility of the system and the healing can’t take place multiple times. Different covalently bonded reversible healing systems are available. They are based on DielsAlder/retro Diels-Alder reaction29, Schiff base reaction30, disulfide exchange reactions

31, 32

etc.

Among these systems, the disulfide based healing systems have several advantages. In this case, the healing takes place at low temperature and under UV-light. The thiyl radicals formed due to the notch (application of mechanical force) can rapidly exchange with the rest of the disulfide bonds and thus healing of the crack. Herein, a unique self-healable antifouling zwitterionic block copolymer (BCP) hydrogel was prepared via a combination of RAFT polymerization and Diels-Alder “click” chemistry. To the best of our knowledge, this is the first report on BCP based hydrogel having antifouling activity and self-healing activity using a combined effect of zwitterionic interaction and disulfide metathesis reaction. The hydrogel was formed by physical interaction between the corecrosslinked (CCL) zwitterionic BCP micelles which consist of PFMA as a hydrophobic core and poly(zwitterion) as a hydrophilic corona. Due to the presence of the poly(zwitterionic) segment, the hydrogel can show a high level of protein repellency property which was examined via a protein adhesion assay using BSA as a model protein. The zwitterionic BCP was synthesized using a sequential copolymerization method where PFMA-RAFT was used as a macro-RAFT agent for the preparation of the second block of poly(N-[3-(dimethylamine)propyl methacrylamide) followed by its betainization using 1,3-propane sultone via a ring opening reaction. The as prepared BCPs were core-crosslinked via DA “click” chemistry using a crosslinker bearing disulfide moiety. It was noted that the hydrogels prepared by interacting CCL zwitterinonic BCPs were able to show a photo-triggered dynamic self-healing behaviour using disulfide metathesis reaction.



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Experimental: Materials and Method: Monomers, methacrylate

like

N-[3-(Dimethylamine)propyl]methacrylamide] (FMA),

RAFT

sulfanylthiocarboxyl)sulfanyl]pentanoic acid

reagent

(DMAPM),

furfuryl

4-cyano-4-[(dodecyl-

(CDTSPA), thermal initiator 4, 4'- Azobis (4-

cyanovaleric acid) (ABCVA) and cystamine dihydrochloride salt, 1,4-dithioerythritol (DTT), 1,3-propane sultone andbovine serum albumin (BSA) were purchased from Sigma-Aldrich, USA. Analytical grade ethanol (EtOH), n-hexane, N, N'-dimethylformamide (DMF), maleic anhydride (MA), sodium acetate (NaOAc), magnesium sulfate (MgSO4), acetic anhydride (Ac2O) were purchased from Merck, India and were used as received.

Synthesis of dithiobismaleimidoethane (DTME), a redox-responsive crosslinker: Cystamine dihydrochloride salt was first neutralized to cystamine using KOH. In a typical formulation, cystamine dihydrochloride (2 g, 8.88 mmol) was suspended in 20 ml of methanol. After the addition of KOH (1.1 g, 19.54 mmol), the white suspension was stirred overnight at room temperature. After 24 h, the white suspension filtered from the solution and methanol was evaporated. The resultant residue was then dissolved in dichloromethane (40 ml) and washed with a saturated solution of NaHCO3 (1 x 10 ml). After that, the dichloromethane phase was dried with MgSO4 followed by evaporation of the solvent using a rota-evaporator to get the pure cystamine. The resultant cystamine (1 g, 6.56 mmol) was then dissolved in 25 ml of acetone. Later maleic anhydride (1.29 g, 13.13 mmol) was added to the mixture. Sudden precipitation of the obtained diacid was observed and the mixture was stirred for next 2 h for completion of the reaction. After that to the reaction mixture, triethylamine (Et3N) (0.5 ml, 6.8 mmol) and NaOAc (7.5 x 10-3, mmol) were added. Then the reaction mixture was heated slowly to reflux temperature with subsequent addition of Ac2O (2 ml, 18.12 mmol). The reaction mixture was then refluxed for four hours and the acetone was evaporated out using a rota-evaporator. The obtained residue was dissolved in DCM (25 ml) and washed with a saturated NaHCO3 solution (13 ml) to remove the formed triethylammonium salt. After that, the DCM phase was dried over MgSO4 and 4 ACS Paragon Plus Environment

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evaporated. The residual Ac2O was removed via azeotropic distillation with cyclohexane. The product was analyzed by FTIR and 1H NMR analyses33. Scheme S1 summarizes the synthesis steps of dithiobismaleimidoethane (DTME). The crude product was purified by column chromatography using a mixed solvent (ethyl acetate: hexane = 1:1) and the obtained product was characterized with 1H NMR using CDCl3 as a solvent. Figure S1 shows the 1H NMR of DTME. The typical resonances appeared at δ = 6.70 ppm (–CH=CH-, H1 and H2), δ = 3.84 ppm (N-CH2–CH2, H3) and δ = 2.92 ppm (–CH2–CH2-S, H4)33. Synthesis of poly(furfuryl methacrylate) (PFMA) via RAFT polymerization: A previously used method was adopted to prepare poly(furfuryl methacrylate) (PFMA)34. GPC analysis showed that the formed polymer had a molecular weight (Mn) and the dispersity (Đ) of 7200 g/mol and 1.34 respectively. Preparation of block copolymer of poly(N-[3-(Dimethylamino)propyl]methacrylamide) (PFMA-b-PDMAPM) using PFMA as a macro-RAFT: In a typical polymerization reaction, PFMA (Mn/GPC = 7200 g mol-1) (0.38 g, 4.85 x 10-2 mmol) was dissolved in 1.2 ml of DMF in a Schlenk tube and subsequently nitrogen was purged into the Schlenk tube for 10 min to make inner atmosphere inert. After that, the monomer, DMAPM (0.5g, 2.425 mmol) and the initiator, ABCVA (0.0035 g, 3.005 x 10-5 mmol) dissolved in 0.5 ml of DMF were injected into the reaction tube. Then the reaction mixture was stirred for 30 min under N2 atmosphere, and it was placed in a preheated oil bath at 80°C. After 24 h, the reaction was terminated by placing the Schlenk tube in an ice bath. Later the whole reaction mixture was precipitated out in diethyl ether. Finally, the BCP, PFMA-b-PDMAPM was dried under vacuum at 50°C for 48 h (For PFMA43-b-PDMAPM72 (Conversion = 97%, Mn/GPC = 19430 g/mol, Đ = 1.38, Mn/NMR= 18830 g/mol; For PFMA43-b-PDMAPM72 (Conversion = 92%, Mn/GPC = 14600 g/mol, Đ = 1.29, Mn/NMR = 13695 g/mol)). Synthesis of zwitterionic block copolymer of PFMA-b-PDMAPM by betainization of PFMA-b-PDMAPM copolymer: The zwitterionic block copolymer (Z-BCP) was prepared via betainization of the PFMA-bPDMAPM. In a typical synthesis method, the PFMA-b-PDMAPM (1 g, 3.172 x 10-3mol) and a 5 ACS Paragon Plus Environment


50 mol% excess (0.58 g, 4.76 x 10-3mol) of 1,3-propane sultone were dissolved in 15 ml of THF, and then the reaction was allowed to continue for 24 h at room temperature. After completion of the reaction, THF was evaporated using a rota-evaporator. The formed zwitterionic BCP was redissolved in a minimum volume of water and then again precipitated out in excess THF. The precipitated material was centrifuged, and the obtained product was dried in a vacuum oven at 30°C. 1H NMR was used to confirm the betainization. 1H NMR analysis was carried out in both d6-DMSO and D2O. Preparation of the redox-responsive core-crosslinked micro-gel via Diels-Alder (DA) reaction: In this reaction, Zwitterionic block copolymer (z-BCP) was crosslinked using a redox-responsive crosslinker, dithiobismaleimidoethane (DTME) via DA reaction. In a typical reaction process, the cross-linker, DTME (0.02 g, 2 wt% with respect toPFMA-b-PDMAPM, BCP) was mixed with 1 g of PFMA-b-PDMAPM in DMSO. Then, water was added (DMSO: water = 1: 4) dropwise in the DMSO solution at constant stirring condition until the DMSO solution becomes turbid followed by the heating of the whole mixture at 60°C for 48 h. The resultant corecrosslinked product was then dialyzed (MWCO 3500 Da) in the presence of DI water to remove the excess DMSO. The final product was obtained after freeze-drying of the product. Protein adsorption assay: The antifouling activity of the synthesized polyzwitterion based BCP was measured by the surface adsorption of the protein over the polymer film. For the preparation of the polymer film, the solution of the BCP (prepared in water: DMSO 4:1 volume mixture) was cast over the glass slide and dried at 50°C under vacuum. Coating of the only homopolymer of PFMA was taken as a control. After that, all the polymer-coated glass slides were incubated separately in 10 ml (2 mg/ml) of BSA solution (prepared in PBS buffer solution having pH of 7.4) for 24 h at 37°C. A certain amount (3 ml) of the BSA solution was withdrawn after the end of the experiment. The change in the absorbance value of the BSA solution was recorded using UV-visible spectrophotometer at a fixed wavelength of 286 nm35, 36. The stability of the coating material was also determined by coating over the different surfaces like- glass, metal and wood. After the application of the coating, all the samples were dipped under water for 5 days and the thickness 6 ACS Paragon Plus Environment

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of the coating over different substrate was measured after drying to observe the stability of the coating material. Self-healing test: The self-healing behaviour of the hydrogel was studied using “scratch & heal” method. Each of the hydrogel (having CCL and non-CCL) was coated over the glass slides and dried under vacuum at 50°C. After that, a notch was created over the hydrogel using an insulin syringe. Then both of the hydrogels were placed in a UV chamber. The healing of the hydrogels was monitored using optical microscopy and AFM analyses. Before placing in the UV chamber, a drop of water was placed over the cut surface. A depth profilometry analysis was carried out in the AFM instrument to determine the efficiency of healing. An optical microscopic analyzer was used to monitor the healing with time sequence. Statistical analysis: The protein adhesion assay was carried out in triplicates and the reported values are the average of all triplicates value ± standard deviation. The student’s t-test was used to compare the statistical significance of test samples against control. The characterization part has been explained in the supporting section. Results and discussions: Scheme 1 represents the preparation of an amphiphilic zwitterionic block copolymer (z-BCP) based on PFMA via RAFT polymerization. In this case, FMA was first polymerized using CDTSPA as a RAFT reagent and ABCVA as an initiator. This PFMA-RAFT reagent was used as a macro-RAFT agent to polymerize DMAPM to prepare PFMA-b-PDMAMP BCP (Scheme 1). The 1H NMR spectrum of PFMA is shown in the supporting section (Figure S2). It shows the characteristic resonances for aromatic protons of the furfuryl group of PFMA at δ = 7.60 ppm, δ = 6.40 ppm and for the methylene protons (-O-CH2) of PFMA unit at δ = 4.90 ppm. Importantly, it shows a particular resonance at δ = 3.28 ppm (designated as “10”) which is due to the –S-CH2group of the CDTSPA RAFT reagent. Figure S3 represents the 1H NMR spectra of the CDTSPA RAFT reagent. This resonance at δ = 3.28 ppm was used to calculate the Mn/NMR = 7478 g/mol which is comparable to the Mn obtained from GPC analysis (Mn/GPC = 7200 g/mol , Ð = 1.34). 7 ACS Paragon Plus Environment


This PFMA was used as a macro-RAFT agent to polymerize DMAPM to prepare BCP, PFMAb-PDMAPM, having different block lengths. The second block of different block lengths was prepared by varying the feed molar ratio of the macro-RAFT (PFMA-RAFT) agent and the monomer N-[3-(Dimethylamino)propyl]methacrylamide (DMAPM) accordingly. Figure 1(i) shows the 1H NMR of the PFMA-b-PDMAPM. The emergence of a new resonance at δ = 3.40 ppm (w.r.t the 1H NMR of PFMA, Figure S2) due to methylene proton of -NH-CH2- (H14), indicates the presence of PDMAPM. Resonances at δ = 4.90 ppm and δ = 3.40 ppm which are due to PFMA and PDMAPM are used to calculate the molar composition of the two blocks as well as the Mn/NMR of the different BCPs. 1H NMR spectra of the synthesized BCP (PFMA-bPDMAPM) was carried out using CDCl3 as a solvent. The molecular weight of the BCP was calculated from the 1H NMR (Mn = 18,834 g/mol) and GPC (Mn = 19,430 g/mol, PDI = 1.38, Figure S4) analyses (carried out using THF as an eluent). The second block having smaller chain length (Mn/NMR = 13,695 g/mol, Mn/GPC = 14,590 g/mol, PDI = 1.29) was also synthesized. Table 1 shows the summary of the preparation of different BCPs. These synthesized BCPs were further modified via betainization of the N,N-dimethyl group (-N(CH3)2) of PDMAPM using 1,3propane sultone via a ring opening reaction (Scheme 1) to prepare the zwitterionic BCP(z-BCPs) of different polyzwitterionic block lengths. The prepared z-BCPs were characterizedby1H NMR analysis performed in both d6-DMSO and D2O as a solvent. In d6-DMSO, resonances for both of the segments of the BCP appeared indicating the formation of open chain morphology (Figure 1(ii)a). Interestingly, it was found that in D2O (Figure 1(ii)b), the resonances for PFMA unit was absent due to the strong attenuation by the polyzwitterionic segments, indicating the formation of “core-shell” structure37, 38. In D2O, PFMA block forms the “core” part, as it is hydrophobic in nature whereas the poly(zwitterionic) segment (hydrophilic in nature) forms the outer shell part. It is reported that in the presence of a thermodynamically friendly solvent, both the segments of the BCP arrange themselves in an unfolded morphology39. In both d6-DMSO and D2O, resonances at δ = 3.62 ppm ((-N+(CH3)2(CH2), H16 and H18), δ = 3.48 ppm (-N+(CH3)2(CH2), H17)and δ = 3.00 ppm (-CH2-SO3-) appeared which indicated the successful betainization of the PDMAPM segment resulting the formation of the poly(zwitterionic) moiety40. The NMR analysis (Figure 1(ii)b) showed that there was complete betainization of the BCP. A complete shift of the resonance peak from δ = 2.25 ppm (-CH3 of PDMAPM, H17, tertiary proton) to δ = 3.25 ppm (-CH3 of PDMAPMZ, H17, quarternary proton) was observed which designates the 8 ACS Paragon Plus Environment

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complete betainization of PDMAPM segment. These polymers were core-crosslinked using dithiobismaleimidoethane (DTME) as a crosslinker via DA reaction between the maleimide functional group of DTME and the furfural group of PFMA bock using DMF as a solvent (Scheme 2). The bifunctional crosslinker, dithiobismaleimidoethane (DTME) was synthesized using a previously used method, as explained in the experimental section. Table 1: Different BCPs via RAFT polymerization using PFMA as a macro-RAFT reagent# in DMF at 80°C.

#

Expt. No.

Polymer Name

Sample Composition

[M]:CTARAFT: [I]

Conv (%)

Mn/NMR (g/mol)

Mn/GPC (g/mol)

PDI

MnTheo (g/mol)

PFMA (mol%)

1

PFPDM1

PFMA43-bPDMAPM72

200 : 4 : 1

95

18834

19430

1.38

20404

38

2

PFPDM2

PFMA43-bPDMAPM 43

100 : 4 : 1

89

13695

14590

1.29

16404

50

RAFT polymerization of DMAPM was carried out using PFMA of Mn = 7200 g/mol as a

Macro-RAFT reagent in DMF at 80°C.

Table 2: Zwitterionic block copolymers (z-BCPs) (PFMA-b-PDMAPMZ) via betainization of PFMA-b-PDMAPM block using 1,3-propane sultone. CMC* (µg/ml)

Expt. No.

Polymer Name

Sample Composition

Mn(g/mol) (considering 100% conversion, calculated from 1H NMR)#

1

PFPDMZ1

PFMA43-b- PDMAPMZ72

27628

29

2

PFPDMZ2

PFMA43-b- PDMAPMZ 43

18947

15

* CMC was calculated from contact angle measurement. #

Mn= Molecular weight of PFMA-b-PDMAPM BCP + (Molecular weight of 1,3-propane

sultone) x DP of PDMAPM block.



Scheme 1: Reaction scheme for the preparation of Zwitterionic BCP.


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Scheme 2: Preparation of a REDOX responsive core-crosslinked (CCL) zwitterionic block copolymer (BCP) using DTME as a REDOX responsive crosslinker.



Figure 1: 1H NMR spectra of (i) poly(furfuryl methacrylate)-b-poly(N-[3(Dimethylamino)propyl]methacrylamide) (PFMA-b-PDMAPM); (ii) betainized PFMA-bPDMAPM in (a) d6-DMSO and in (b) D2O.

Dithiobismaleimidoethane (DTME) and all the BCPs were further characterized by FTIR analysis. Figure S5a represents the FTIR spectrum of cystamine and modified DTME. 12 ACS Paragon Plus Environment

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Cystamine shows characteristic absorption peaks at 3364 cm-1 and 3288 cm-1 due to the presence of –NH stretching of diamine group and at the 470 cm-1 for S-S bond respectively41. After modification with maleic anhydride, the absorption band for the –NH stretching diminished and a new peak appeared at 1172 cm-1, 1393 cm-1 for C-N stretching, 1705 cm-1 for carbonyl (>C=O) of cyclic maleimide ring and 1635 cm-1 for a C=C bond of maleimide ring42. From the Figure S5b, it is observed that the PFMA shows characteristic absorption band at 2925 cm-1, 1110 cm-1 and 1024 cm-1 which are due to the presence of –CH2 stretching, C-O-C stretching and furan ring stretching respectively. In case of PFMA-b-PDMAPM, characteristic absorption band for PDMAMPM group has been appeared at 2925 cm-1-2840 cm-1, 1732 cm-1, 1431 cm-1 which are due to the asymmetric and symmetric stretching of –CH2 group, stretching of >C=O group and C-N stretching respectively42. After betainization of the PFMA-b-PDMAPM, the resultant polyzwitterionic BCP shows characteristic absorption band at 1033 cm-1 and 1193 cm-1 due to the symmetric and antisymmetric vibrational stretching of SO3- group43 respectively. It also shows absorption band at 1477 cm-1 and 931 cm-1 which are attributed to the -CH3 bending vibration of [RN(CH3)3]+ and -CH3 stretching vibration of [RN(CH3)3]+ respectively. This indicates the successful betainization of the PDMAPM group in PFMA-b-PDMAPM. The prepared zwitterionic BCP was further core-crosslinked using a REDOX responsive crosslinker. The formation of the DA product was confirmed via monitoring a relative decrease in the absorption band at 1016 cm-1 which is due to the furan ring stretching and subsequent increase in the intensity of the absorption band at 1655 cm-1, indicating the formation of >C=C< due to DA reaction. Analysis of solution property and self-assembly of the synthesized zwitterionic block copolymers: Amphiphilic BCPs having ionic segment are a fascinating class of macromolecules due to their wide and interesting range of self-assembled structure depending on the nature of the solvent. These types of ionic macromolecules can adopt a plethora of morphologies including cylindrical micelles, spherical micelles, lamellae, vesicle etc.44. In this work, we have followed a very convenient preparation method of the polymeric micelle. It is known that an Am-BCP undergoes micellization when it is dissolved in a selective solvent at a constant temperature and certain concentration (CMC= critical micelle concentration). Here, the zwitterionic BCP is dissolved in DMSO which is a thermodynamically favourable solvent for both the segments. So in the 13 ACS Paragon Plus Environment


presence of DMSO, the BCP acts as a unimer. After that water, a selective solvent for the polyzwitterionic segment and a non-solvent for the rest of the segment was added dropwise to form the micelle. Upon addition of the water, the BCP conformed itself in a nano scopicmicellar aggregates having a spherical morphology. The spherical micelle hasa well-defined hydrophobic core formed by the hydrophobic segment (PFMA) surrounded by the soft corona of polyzwitterion. This process of micellization is very much analogous to the micellization of the low molecular weight surfactant. It is reported that the self-assembly of the BCP occurs to minimize the energetically unfavourable solvophobic interactions. To get rid of the solvophobic interactions the hydrophobic segment undergoes aggregation (forming the core) whereas the hydrophilic segment interacts with the friendly solvent to stabilize the micelle. For monitoring the disparity of the CMC value, the block length of the zwitterionic BCP (hydrophilic segment) has been varied significantly (DP = 43 and 72). CMC value was determined using goniometer. It was observed that with increase in the block length of the polyzwitterionic segment, the respective CMC value increased significantly as summarized in Table 2. The formed micelle of the BCP (PFMA43-b- PDMAPMZ72) had a size of 298 ± 5 nm and particle distribution index (PDI) of 0.396, determined by DLS analysis using water as solvent (Figure 2a). Upon corecrosslinking (CCL), the size of the micelle reduced to 258 ± 5 nm (PDI = 0.302) (Figure 2b). In each case, the concentration of the BCP solution was maintained at 2 mg/ml. It was observed that the CCL micelle shows a higher diameter (300 ± 5 nm, PDI = 0.320) in the presence of the NaCl solution having a concentration of 0.154 M. The expansion of the micellar size is mainly due to the “antipolyelectrolyte” effect of poly(zwitterionic) segment. The probable mechanism behind the change in the micellar size is schematically represented in the same figure (Figure 2). From the PDI value of the respective spherical micelle in water and the salt solution, it can be concluded that the formed micelle was stable in solution. A relatively higher PDI value of the micelle in water compared to the salt solution supports the self-interaction of the zwitterionic segment of BCP in water. Whereas, in a salt solution, the Na+ and Cl- provide a screening effect to the zwitterionic segments that resist the formation of nano to micro-aggregates. The size of the CCL micelle was also observedin the presence of aqueous and salt (0.154 M NaCl) solution. The Figure 2d and Figure 2e, indicate that in the presence of DTT the size of the micelle increased; (in water = 286 nm, PDI of 0.415; in salt solution = 332 nm, PDI of 0.398). This supports the reduction of the REDOX responsive disulfidic core (-S-S-) and formation of the free thiol (14 ACS Paragon Plus Environment

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SH)45-48. The probable mechanism behind the reduction process is schematically represented in Figure 2. The behaviour of the CCL zwitterionic micelle in the presence of the salt solution of varying concentration was also observed. In pure water, the micelle size of the polyzwitterionic BCP was in the range of 258 ± 5 nm having a PDI of 0.302. As described earlier that the interaction between the ammonium cation ((CH3)2N+-) and the sulfonate anion (-SO3-), results in nano to microscopic aggregates leading to higher PDI.



Figure 2: DLS analysis of (a) PFMA-b-poly(sulfobetaine), (b) CCL PFMA-b-poly(sulfobetaine) in water, (c) CCL PFMA-b-poly(sulfobetaine) in salt solution, (d) CCL PFMA-bpoly(sulfobetaine) in aqueous solution of DTT, (e) CCL PFMA-b-poly(sulfobetaine) in salt solution. It is reported that the polysulfobetainemethylacrylate can form a physical hydrogel at a concentration of 61% using inter and intrachain Coulombic interaction between the charged 16 ACS Paragon Plus Environment

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segments. It was observed that with an increase in the salt concentration the size of the micelle also increased upto a certain limit. It is a well-known fact that the presence of NaCl can screen the electrostatic interaction between the polyelectrolyte segments leading to the disruption of inter and intrachain Coulombic attraction49, 50 After a critical concentration of 0.2 M of NaCl solution, the size of the micelle again decreased with a subsequent decrease in the PDI value (0.265 at 0.5 M NaCl concentration). After that again the drop in the size of the micelle was observed at a salt concentration of 2 M (PDI = 0.281) and 3M (PDI = 0.251) (Figure 3a). The probable effect of the salt over the typical behaviour of the polyzwitterionic Am-BCP is schematically represented in Figure 3b. At lower concentration of salt (0.154 M), a gelling effect was observed.This is due to the dynamic equilibrium between the electrostatic interaction between the polyzwitterionic segment and the shielding effect of the Na+ and Cl- counterions. However, at a very high concentration of the salt (3 M), the reduction in the size of the micelle was due to the predominant shielding effect of the salt ions over the Coulombic attraction present in between the polyzwitterionic segments. The image regarding the behaviour of BCP at different salt concentration has been represented in Figure 3b.



Figure 3: (a) Variation of the size of the zwitterionic BCP with the variation of the NaCl solution concentration and (b) probable structure of the BCP in the presence of NaCl solution having different concentrations.


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Morphological analysis: Now-a-days, Am-BCP micelle with a polyelectrolyte block has attracted a great attention due to the change in the behaviour of polyelectrolyte corona in the presence of salt concentration, solvophilic interaction, pH etc. Here, we have carried out FESEM, HRTEM and AFM analyses of the formed zwitterionic Am-BCP to get an idea about the morphology of the formed micelle. Figure 4a represents the surface morphology of the crosslinked PFMA homopolymer; whereas Figure 4band Figure 4c show the FESEM images of the formed polyzwitterionic micelle and the CCL micelle. It is observed that the Am-BCPs have oriented themselves in a spherical micellar structure. When the polyzwitterionic micelles aremixed at a very high concentration (200 mg/ml), it forms a gel via Coulombic interaction. Figure 4d shows the morphology of the gel formed by the polyzwitterionic BCP. The morphology obtained from the HRTEM analysis also corroborates the morphology acquired from FESEM analysis. A beautiful “core-shell” structure was obtained where the hydrophobic PFMA segment formed the core (a darker portion of the micelle) whereas the corona (lighter dark portion) of the micelle formed by the polyzwitterionic segment (Figure 5a and Figure 5b). The presence of the ionic interaction was also evidenced from the HRTEM analysis, (Figure 5c) where it was nicely observed that at higher concentration (2 mg/ml), the zwitterionic BCPs had started aggregating with each other leading to bigger particle size.Xie et.al.51 reported the interaction zwitterionic phosphorylcholine polymers via aggregation-induced emission (AIE) feature and Lucas et.al.52also reported the amphiphilic micellar interactions through liquid-cell transmission electron microscopy (LCTEM) study. As discussed in the DLS analysis, upon core-crosslinking the size of the micelle decreased. The same observation was also observed in the HRTEM analysis. A reduction in size was observed from (125 ± 5) nm to (104 ± 5) nm upon core-crosslinking of the micelle. The formation of the “core-shell” type of morphology is the result of the two parameters- i. change in the molecular curvature of the micelle due to the variation in the chain length of the soluble and the insoluble domain of the respective BCP and ii. dynamic equilibrium between the solvophilic and the solvophobic interactions by the hydrophilic and the hydrophobic segments respectively53. The balance between these solvophobic and the solvophilic interactions generates an optimal surface area“Ac”at the interface of the hydrophobic and hydrophilic segment. This area together with the length (Lc) and the volume (Vc) of the hydrophobic segment generates the “packing parameter (P)”54. This can be expressed as19 ACS Paragon Plus Environment


.

Packing parameter is the ratio of the volume occupied by the hydrophobic block to the total volume occupied by the copolymer. It is reported that if the value of “P” is ≤ 1/3, the micelle arranged itself in a spherical type of morphology as in our case. AFM analysis of the respective BCP micelle and the CCL BCP micelle is represented in Figure S6. AFM analysis also supports the formation of spherical type micelle by the Am-BCP.

Figure 4: FESEM images of (a) PFMA homopolymer, (b) PFMA-b-poly(sulfobetaine), (c) CCL PFMA-b-poly(sulfobetaine) and (d) hydrogel formed by CCL PFMA-b-poly(sulfobetaine).


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Figure 5: HRTEM images of (a) CCL PFMA-b-poly(sulfobetaine), (b) PFMA-bpoly(sulfobetaine) and (c) self-assembled zwitterionic BCP (concentration = 2 mg/ml). The formation of the amphiphilic block copolymer can also be evidencedby the water contact angle analysis. The obtained results have been explained in the supporting information section (Figure S7). SAXS analysis: The SAXS is a powerful tool to analyze the nano scale heterogeneity present in a hydrogel system 55, 56. In this investigation, we used this tool to analyze the microphase separation present in the zwitterionically interacted hydrogel. The SAXS profiles of the crosslinked PFMA homopolymer and CCL polyzwitterionic BCP are shown in Figure S8(a) and Figure S8(b) respectively. It was observed that a higher scattering was observed in case of the zwitterionic BCP as compared to the crosslinked PFMA homopolymer. The Power law was utilized to analyze the SAXS profile of the self-aggregated zwitterionic block copolymeric hydrogel within the small “q” region57: I(q) = I(0)q-p



Here, I(0) is the prefactor, p is related to the fractal dimension (D). p can be related to either the mass fractal dimension (Dm) or the surfacefractal dimension (Ds): p = Dm (p < 3, mass fractal) p = 6 –Ds(3 < p < 4, surface fractal) The agglomeration of the nanoparticles inside a matrix is a result of the scaling, self-similarity and the universality58. The self-similarity can be expressed as fractals which can be evaluated by inter-cluster aggregation and diffusion-limited agglomeration models. The openness of the hydrogel structure can be indicated by ‘Dm’ which is ‘1’ for the loosely bound aggregates and ‘3’ for the dense aggregates. On the other hand ‘Ds’ which is the surface fractal is ‘3’ for the rough surface and ‘2’ for the smooth surface57. The scattering from the fractal surface can be determined by using the Power law where the exponent ‘p’was calculated from the slope of the .lnI(q)–ln

q plots.

From the Figure S7(a) and Figure S7(b), it can be observed that the correlation coefficient (R) value is more than 0.98. As shown in Figure S7(a) that for the crosslinked PFMA homopolymer the ‘p’has a value of 2.19, whereas in case of the core-crosslinked zwitterionic BCP the obtained ‘p’ value is 3.10. This indicates the formation of denser aggregates in case of the zwitterionic block copolymer. This phenomenon strongly supports the formation of the ionic aggregates. It also indicates the formation of the rough surface due to the generation of the ionic aggregates as also observed from the FESEM image (Figure 4d). Thermal analysis: DSC analysis was carried out to determine the thermoreversible behaviour of the Am-BCP hydrogel formed by the Coulombic interaction between the ammonium cation ((CH3)2-N+-) and the sulfonate anion (-SO3-) of the polyzwitterionic segment. The CCL BCP based self-assembled hydrogel was heated from –100°C to +200°C using a heating rate of 20°C/ min. Figure 6 shows the DSC traces of the hydrogel, and from the result, it can be observed that a broad endotherm appeared at 165°C which stands for the rDA reaction. In the cooling curve, a broad exotherm was observed at 65°C (enlarged picture is shown in inset) which indicated the reformation of the DA product between PFMA and the DTME via [4 + 2] cycloaddition reaction. The appearance 22 ACS Paragon Plus Environment

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of the weak exotherm is due to the fast cooling process that hinders the completion of the cycloaddition reaction. Several authors reported this type of observation. The glass transition (Tg) value of the individual segment of the Am-zwitterionic BCP was also determined from the DSC analysis. It was found that the PFMA segment showed a Tg of 35°C whereas the poly(zwitterionic) segment showed Tg at 93°C. The higher Tg value obtained in case of the poly(zwitterionic) segment is due to the formation of the ionic cluster between the cationic ((CH3)2-N+-) and anionic (-SO3-) part that restricts the segmental movement of the respective block59. The DSC analysis of the BCP having non-crosslinked core was also carried out as a control experiment, and it was observed that no significant change was observed near 165°C during heating and at 65°C during the cooling cycle.

Figure 6: DSC traces of PFMA-b-poly(sulfobetaine),with and without CCL. Self-healing study: In case of our self-assembled zwitterionic hydrogel, the self-healing experiment was carried out using a combined effect of conventional photo-induced radical disulfide metathesis (PRDM) reaction and the ionic interaction between the zwitterionic segments. The as prepared zwitterionic hydrogel was coated over the glass slide and dried subsequently. The DTME crosslinked PFMA was used as a control in the self-healing experiment. The zwitterionic hydrogel having no DTME crosslinker was also used to monitor the impact of disulfide exchange reaction over the self-healing. A small notch was generated over each of the substrate using an 23 ACS Paragon Plus Environment


insulin syringe. After that, all the samples were placed in the UV chamber. A drop of water was placed over the cut surface of each sample. Healing of the hydrogels was monitored via optical microscopy and AFM depth profilometry analyses. Figure 7 represents the fate of the notched samples upon exposure to UV light for two hours. It was observed that notch over the DTME crosslinked PFMA remains the same after the prolonged exposure to the UV light (after 120 min (t120)) (Figure 7a and Figure 7(a1)). Whereas, a zwitterionic hydrogel containing BCP having no CCL and with CCL showed self-healing behaviour. In the presence of water, the selfassembled hydrogel having no DTME crosslinked BCP showed self-healing behaviour (Figure 7b and Figure 7(b1) but the extent of the self-healing is comparatively lower than the corecrosslinked one (Figure 7c and Figure 7(c1)). In the second case, the self-healing occurs due to the zwitterionic interaction of the BCP in the presence of the water.

Figure 7: Self-healing study of notched PFMA (a and a1), BCP hydrogel without CCL (b and b1) and BCP hydrogel having CCL (c and c1) via optical microscopy after exposure to UV light at t = 0 min and t = 120 min.

Water provides a drooping effect to the BCP segment that helped in the formation of the ionic entanglement60, 61. The hydrogel consists of DTME crosslinked BCP showed complete healing which might be due to the synergistic effect of zwitterionic interaction and the disulfide metathesis reaction. In the presence of the UV light, the disulfide bond was homolytically cleaved to generate sulfenyl radical as observed in the Scheme 3. The exchange of this sulfenyl


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radical with the uncleaved disulfide bond accelerates the healing process62. This reshuffling of the disulfide bonds in the polymeric network leads to the self-healing of the notched hydrogel.

Scheme 3: Schematic representation of the healing process of a notched hydrogel in the presence of UV light via photo-triggered disulfide metathesis reaction.



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To confirm the synergistic contribution of zwitterionic interaction and the disulfide metathesis reaction over the self-healing effect, we have carried out the AFM depth profilometry analysis of all the coating compositions (Figure 8). Interestingly, it has been found that as compared to the other two coating compositions having disulfide crosslinked PFMA (Figure 8(a) and Figure 8(a1)) (healing efficiency = 15%) and PFMA-b-poly(sulfobetaine) (Figure 8(b) and Figure 8(b1)) (healing efficiency = 36%), the decrease in the depth height in case of the coating containing both, poly(zwitterion) and disulfide core crosslink is maximum ((Figure 8(c) and Figure 8(c1)) (healing efficiency = 88%). Healing efficiency has been determined using the following equationHealing efficiency (%) =

100

Figure 8: Self-healing study of notched PFMA (a and a1), z-BCP hydrogel without CCL (b and b1) and z-BCP hydrogel having CCL (c anda c1) via AFM depth profilometry study after exposure to UV light at t = 0 min and t = 120 min. (a, b and c are 2D topographic images and a1, b1 and c1 are 3D topographic images with depth analysis).


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The mechanical property of the self-healed hydrogel was analyzed with nano-indentation test and the discussion against the obtained result has been included in the supporting information (Table S1). Protein adhesion assay: The protein resistant property of the prepared polyzwitterionic hydrogel was evaluated by the BSA adsorption study over the hydrogel surface. FESEM analysis was used to observe the adhesion of the BSA protein visually. Here, PFMA homopolymer acted as a control (Figure 9a). It was observed that the presence of the polyzwitterionic unit strongly resists the adhesion of BSA compared to the PFMA homopolymer surface (Figure 9b). This phenomenonis attributed to the high hydration efficiency of the polyzwitterionic segment leading to the formation of a strong water shielding layer over the BCP surface. The formation of the shielding layer inhibits the biofilm formation over the hydrogel surface63, 64. The protein adhesion was also assessed in the presence of the salt solution (0.154 M NaCl solution). It was observed that in the presence of salt solution the protein repellency property of the hydrogel was getting improved (Figure 9c). This is mainly due to the antipolyelectrolyte effect of the polyzwitterionic segment65, 66. As discussed earlier due to the antipolyelectrolyte effect, the polyzwitterionic segment arranged them in an unfolded brush-likemanner in the presence of the salt solution. This brush-like morphology helps to generate water-shielded surface which helps them to resist the biofilm formation. The respective working action of the PFMA homopolymer and Am-BCP is schematically represented in Figure 9. Protein adhesion was also studied using UV-visible study (Figure 9d). As described in the experimental section that the PFMA homopolymer and the hydrogel-coated glass slides were dipped into the BSA solution having a concentration of 2 mg/ml for 24 h at 37°C temperature. After that 1 ml of BSA solution was withdrawn from each setup and the change in absorbance value of the solution at λmax = 286 nm (characteristic absorbance value for BSA protein) was monitored to have an idea about the adherence of the BSA protein over the polymeric surface.



Figure 9: Protein adsorption study over (a) PFMA, (b) water swollen CCL PFMA-bpoly(sulfobetaine) hydrogel, (c) salt solution swollen CCL PFMA-b-poly(sulfobetaine) hydrogel, (d) BSA protein adsorption study via UV-vis study and (e) graphical representation of comparative protein adsorption over mentioned components.

It was observed that the absorbance value of BSA protein drastically reduced in the presence of the PFMA homopolymer which designates the maximum adsorption of the BSA protein over the PFMA surface, as observed through the FESEM study (Figure 9a). On the other hand, Am-zwitterionic BCP prevents the protein adhesion significantly. So the absorbance of the BSA solution increased compared to the BSA solution withdrawn from PFMA set up (p < 0.05). Interestingly, it was noticed that when a salt solution of BSA was used instead of the water solution of BSA, the adsorption of the protein reduced significantly (p < 0.05) which is due to the antipolyelectrolyte effect as explained earlier. This phenomenon results in a higher absorbance value which is much similar to the absorbance of the pure BSA solution67. The 28 ACS Paragon Plus Environment

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amount of the BSA adsorption over the PFMA and the BCP surface is graphically represented in Figure 9e. To get an idea about the stability of the coating material over the different surfaces like glass, metal (aluminium) and wood upon application of water, BCP based hydrogel was coated over the as mentioned surfaces. After that, all the systems have been dipped into the water for 5 days followed by drying of the substrate at 50°C. Figure S9 shows the images of the coating after the treatment with water. The initial and the final (after water treatment) thickness of the coating were found to be nearly the same, and the obtained data has been summarized in Table S2. Conclusion: In conclusion, a self-healable antifouling hydrogel material was developed via RAFT polymerization and DA “click” chemistry. The synthesized hydrogel was the result of the ionically interacted CCL zwitterionic BCP having poly(furfuryl methacrylate) (PFMA) as coreforming

block

and

poly([Dimethyl-[3-(2-methyl-acryloylamino)-propyl]-(3-

sulfopropyl)ammonium) (poly(sulfobetaine), PSB) as a shell. The core of the BCP was crosslinked with DTME, a REDOX responsive crosslinker. It was observed that the zwitterionic hydrogel having CCL Am-BCP showed self-healing behaviour utilizing a synergistic effect of photo-triggered dynamic disulfide metathesis reaction and zwitterionic interaction. The selfhealing was monitored via optical microscopy, AFM depth profilometry and depth sensing nanoindentation study (NINT). Moreover, the hydrogel was able to inhibit the biofilm formation over the substrate surface (antifouling activity). This new class of multifunctional self-healable hydrogel may have potential application in designing smart polymeric material. Supporting Information Characterizations procedures, a scheme for the preparation of dithiobismaleimidoethane (DTME), characterization data (NMR, FTIR, AFM, GPC, contact angle, nano-indentation and SAXS analyses) for compounds in this manuscript, images for coating stability study and Table for durability test of the coating.



Author Information Corresponding Author Email- [email protected]; [email protected] Conflicts of interest There are no conflicts to declare. Acknowledgements IIT Kharagpur kindly provided funding for Mr. Sovan Lal Banerjee's fellowship. References: 1.

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Self-Healable Antifouling Zwitterionic Hydrogel Based on Synergistic

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