proteins nanocomposites with enhanced

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Materials and Interfaces

Waterborne hybrid acrylic/proteins nanocomposites with enhanced hydrophobicity by incorporating a water repelling protein Mariana Allasia, Mario Cesar Guillermo Passeggi (Jr.), Luis M. Gugliotta, and Roque J. Minari Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.9b02518 • Publication Date (Web): 13 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

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Industrial & Engineering Chemistry Research

Waterborne hybrid acrylic/proteins nanocomposites with enhanced hydrophobicity by incorporating a water repelling protein Mariana Allasia‡, Mario C.G. Passeggi(Jr.)§,⸸, Luis M. Gugliotta‡,⸸, Roque J. Minari*‡,⸸

‡Polymer

Reaction Engineering Group, INTEC (Universidad Nacional del

Litoral-CONICET), Güemes 3450, Santa Fe 3000, Argentina.

§Physics

of Surfaces and Interfaces Laboratory, IFIS Litoral (Universidad

Nacional del Litoral-CONICET), Güemes 3450, Santa Fe 3000, Argentina.

⸸Facultad

de Ingeniería Química (Universidad Nacional del Litoral), Santiago

del Estero 2829, Santa Fe 3000, Argentina.

*E-mail: [email protected]

1 ACS Paragon Plus Environment

Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Keywords: Miniemulsion Polymerization, Acrylic/Protein Nanocomposites, Ecofriendly Latex, Water Resistance, Surface Hydrophobicity.

Abstract

The incorporation of natural proteins as renewable resource in the production of hybrid latexes has high interest in academia and industry, due to the opportunity to synthesize novel eco-friendly materials with special functionalities, improved properties and biodegradable character. This article pursues the synthesis by miniemulsion

polymerization

of

novel

waterborne

nanoparticles

which

adequately combine 3 main components: i) a low Tg acrylic copolymer that provides controllable mechanical properties; ii) casein, an amphiphilic protein that allows polymer particle stabilization without employing an emulsifier, which means a challenge for producing stable polymer dispersions; and iii) zein, a water repelling protein which balances the hydrophilic character of casein. The


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obtained emulsifier-free hybrid latexes present high compatibility among components, resulting in new materials with improved properties. Hybrid films formed exhibit enhanced water resistance when incorporating a very low fraction of zein (5 % with respect to the total protein content), and a hydrophobic surface with contact angle similar to the pure acrylic film (82°). Main characteristics of hybrid nanoparticles and relevant properties of obtained films are here discussed.



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Introduction During the last decade, the substitution of petroleum-based polymers by renewable and more sustainable materials, and the development of industrially applicable polymers from biobased feedstocks have been the motivation of many investigations.1–3Natural proteins are promising biopolymers for partially substituting petroleum-based polymers, due to their superior biodegradability, edibility, low toxicity, and convenient absorbability.4 In addition, proteins contain functionalities

like amine and carboxyl, which provide opportunities for

introducing structure modification.5,6Their main applications are in the area of paper coatings, adhesives, paint binders,7food packaging and controlled release of additives and bioactive compounds.8 Casein, a natural protein present in milk, was used in film formation applications. However, pure casein films present low resistance to microbial attack and wet rub, and have a low capability of deformation without failure. In this scenario, the synergetic combination of a natural protein with a high Tg, as casein, with a soft acrylic polymer is a promising system to obtain hybrid materials with improved mechanical behavior. In this regard, the synthesis of


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casein-stabilized latexes by free-emulsifier emulsion polymerization using persulfate initiators has been previously investigated.9 Li et al.10 synthesize acrylic/casein latexes by emulsion polymerization, where the grafting of the acrylic polymer onto casein chains was promoted by the redox initiation between an alkyl hydroperoxide and casein amine groups. According to Picchio et al.,11,12such redox initiation allowed the production of acrylic/casein particles with high degree of grafting (i.e., compatibility) when a low protein concentration is used. On the other hand, the hydrophilic character of casein and the low grafting degree reached when a high biomaterial content was used, resulted in hybrid films with poor water resistance. Therefore, the use of a (metha)acrylated casein appeared as an interestly strategy for producing hybrid latexes with high biomaterial content and enhanced degree of compatibilization between both components.6,7 However, high degrees of casein modification were required to improve the organic solvent permeability and water resistance of films, thus affecting their mechanical properties.13 Zein, a prolamine of corn, is a water insoluble protein with a low nutritional value due to it lacks of the essential amino acids lysine and tryptophan. It is 5 ACS Paragon Plus Environment


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obtained as a byproduct from corn processing during the production of bioethanol and corn meal by wet-milling process.14,15 Zein has awaken a high interest in food and pharmaceutical applications due to its film forming ability.16It has been used in encapsulation,17as drug delivery base,18and in tissue scaffolding.19 Pure zein films present exceptional characteristics as tough, glossy, hydrophobic, grease proof, and resistant to microbial attack,14,20 but they are usually highly brittle, requiring plasticization to enhance their mechanical performance.4,21 This work pursues the synthesis of waterborne hybrid acrylic/protein nanocomposites with an adequate balance between an amphiphilic biomaterial (casein), that stabilizes acrylic polymer particles and a water insoluble protein (zein), which provides a water repelling characteristic. To this effect, miniemulsion polymerization was employed for obtaining emulsifier-free acrylic latexes containing casein and zein as natural biomaterials. Following this strategy, latexes with high protein concentration (25% weight based on monomers) and moderate/high solids content (35 wt%) were synthesized. Miniemulsion polymerization appears as an alternative for synthesizing hybrid 6 ACS Paragon Plus Environment

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latexes enabling the incorporation of hydrophobic components into the monomer droplets (50-500 nm), which are directly nucleated and transformed in the polymer particles.22,23 This proposal demonstrates that it is possible to improve the water resistance of casein-based latexes by incorporating a hydrophobic protein as zein. The performance of the produced hybrid materials, containing different concentration of proteins, were assessed in terms of film properties, paying special attention on water resistance and surface hydrophobicity. As the authors are aware, the synthesis of emulsifier-free polymer particles by miniemulsion polymerization, in which casein acts as stabilizer, has not previously reported. 1. Materials and methods Both employed natural proteins, casein from bovine milk (provided by Tecnicom SRL) and zein (Sigma), were technical grade. Methyl methacrylate (MMA) and butyl acrylate (BA) monomers containing traces of the inhibitor mono methyl ether hydroquinone (Aldrich) and glycidyl methacrylate (GMA, Aldrich) as casein functionalizing agent were used. The employed initiators were tert-butyl hydroperoxide (TBHP, Aldrich) and potassium persulfate (KPS,



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Mallinckrodt). Other reagents were sodium lauryl sulphate (SLS, Cicarelli, 95% purity) and Dowfax 2EP (Dow, solution containing 45% of dodecyl diphenyl ether) as surfactants, sodium carbonate (Na2CO3, Cicarelli) as buffer, octadecyl acrylate (OA Sigma-Aldrich, 97% purity) as costabilizer, and methyl ethyl ketone (MEK, Anedra) and tetrahydrofuran (THF, Cicarelli) as organic solvents. Microscopy grade uranyl acetate 1 wt% solution (UAc, EMS) and formvar (polyvinyl formal, Fluka) were employed for TEM samples preparation. All the reagents were used directly as received, without any further purification. In addition, distilled and deionized water was used throughout this work. 1.1.

Polymerization process

The codes of latexes and films contain the information of their composition by the abbreviations “A”, for acrylics monomers, “CN or CF”, for native or functionalized casein, respectively, and “Z”, for zein. Thus, A/CNZ refers to a sample containing acrylics, native casein and zein. A protein-free latex was run as a reference (experiment code A). In this case, Dowfax 2EP was employed as emulsifier.

Miniemulsification 8 ACS Paragon Plus Environment

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The following characteristics were constant in all the experiments: a) 35 wt% of solids content; b) 25 %wbm (weight based on monomer) of total protein (except for the reference protein-free miniemulsion); and c) 4% wbm of OA as costabilizer. To obtain emulsifier-free hybrid latexes, natural proteins (casein and zein) in their native state as well as methacrylated casein were used (Table 1). Functionalized casein with 8 methacrylic groups per molecule of protein was obtained according to the procedure described in Picchio et al.,6where glycidyl methacrylate (GMA) was used as the functionalization reagent. This modification was carried out before the miniemulsification stage. In latexes where native casein was used, GMA was incorporated in the organic phase with the same GMA/casein ratio used in casein methacrylation. Miniemulsions were prepared by slowly adding the organic phase including zein, acrylic monomers (with a BA/MMA ratio of 80/20) and the costabilizer OA (4 %wbm) during 20 min, and applying ultrasound to the aqueous phase that contained dissolved Na2CO3 (0.4 weight based on water phase) and casein (native or functionalized) as oil droplets stabilizer. Then, dispersions were 9 ACS Paragon Plus Environment


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sonicated for additional 20 minutes. Miniemulsifications were performed in a cooled jacketed vessel with permanent magnetic stirring and controlled temperature below 30 °C. A ultrasonic processor, Sonics VC 750 (power 750 watts), operated at 70% of amplitude and with cycles of 10 seconds on and 5 off was used.

Polymerization Polymerizations were carried out in batch in a 100 mL glass reactor equipped with a reflux condenser and under nitrogen bubbling. Reaction temperature was kept constant (80 °C) by adjusting the temperature of the fluid in the reactor jacket with a thermostactic bath. Two initiators were employed (Table 1): KPS (0.8 %wbm) and TBHP (0.24 %wbm). Notice that both initiators produce radicals in the aqueous phase; however TBHP is part of a redox initiation system that also involves the amine groups of protein, according to Li et al.10 Table 1. Formulation of the synthesized hybrid latexes.

Experime

Protein (% wbtpa)

Total

Initiator Emulsifie 10

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nt

Native

Modified

Native

protein

casein

casein

Zein

(% wbmb)

0

0

0

0

KPS

Dowfax

A/CN

100

0

0

25

TBHP

-

A/CF

0

100

0

25

TBHP

-

A/CNZ

95

0

5

25

TBHP

-

A/CFZ

0

95

5

25

KPS

-

A

r

(a) wbtp: weight based on total protein; (b) wbm: weight based on monomer.

1.2.

Miniemulsion and latex characterization

The overall monomer conversion (x) was measured by gravimetry and proteins were not considered for calculating x. Average droplets diameter (dd) and particles diameter (dp) were determined by dynamic light scattering (DLS) employing a BI-9000 photometer from Brookhaven . To avoid droplets destabilization and the monomers loss from droplets and particles during DLS characterization, samples were prepared by diluting latexes in a water solution saturated with casein (or Dowfax 2EP for experiment A) and monomers. The percentage of protein chemically linked to the acrylic polymer (protein grafting efficiency, PGE) was calculated as the mass ratio between the grafted protein and the total loaded protein.6 Ungrafted protein was separated from



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latexes by centrifugation and redispersion with deionized water and a SLS solution. Then, ungrafted protein contained in latex supernatants was quantified by means of UV spectroscopy using a Perkin-Elmer Lambda 25 UV-Visible spectrophotometer. Soluble protein concentration was calculated by correlating the area of peak at 280 nm with a calibration of protein concentration. Grafted protein mass resulted from the difference between the loaded and the nonlinked protein. The fraction of insoluble acrylic (IF) contained in the hybrid materials was determined by soxhlet extraction with THF for 24 h. It is worth noticing that IF could be constituted by the acrylic gel, the grafted acrylic-protein copolymer, and the free protein, which is also insoluble in THF. Particles morphology was investigated by transmission electron microscopy (TEM), employing a JEOL 100 CX(100 kV) microscope. TEM samples were prepared by placing a droplet of diluted latex (around 0.01 wt % of solids content) on a formvar coated copper grid and drying it a room temperature. Then, a drop of 1 wt % UAc solution was added on it to negative stain solid surface of particles sample. 12 ACS Paragon Plus Environment

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1.3.

Film characterization

The hybrid films were obtained by casting the latexes and drying them at 22 °C and 55% of RH over a week. Minimum film formation temperature (MFFT) of acrylic-proteins latexes was measured with an optical method previously reported elsewhere.24 A film (120 µm thickness) was casted on a large metal table with a temperature gradient. Then, the MFFT value was considered as the minimum temperature on the metal table, where the film was judged to be clear. Atomic Force Microscopy (AFM) in tapping mode was employed to determine the film morphology of the hybrid nanocomposites. A commercial Nanotec Electronic equipment and All-In-One-Al silicon cantilevers (Budget Sensors) with a nominal spring constant k=40 N/m and a resonance frequency of 350 kHz were employed. For surface imaging, latexes were cast onto seal paper (120 m wet-thickness) and dried at room temperature overnight. For crosssection AFM imaging, cast films of about 1 mm of thickness were transversally cut after freezing with liquid nitrogen. Acquisition and images processing were carried out with a WS×M free software.25



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Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were carried out with Q2000 and Q500 equipments (TA Instruments), respectively. TGA was performed onto sample films of 5 mg heated from 25 to 600 °C, at a heating rate of 10 °C/min and under nitrogen atmosphere. The maximal decomposition temperature (Td,max) was determined from the derivative weight loss curve as the temperature at maximum of the main peak. Due to both proteins showed glass transitions (at around 170 °C) close to the beginning of their thermal decomposition (see Figure S.1 of Supporting Information, SI),26,27 DSC were carried out from −80 to 130 °C at a heating rate of 10 °C/min. Film samples (of about 5 mg) were assessed twice and its glass transition temperature (Tg) was determined during the second run as the midpoint temperature of the observed heat capacity change. Face-to-face blocking resistance of the films was evaluated following ASTM D 4946-89. To this effect, latexes were applied on sealed paper using a frame applicator with a wet thickness of 120 µm and dried during two days at room temperature. Then, six film squares were cut with an area of 38 mm × 38 mm and placed with the film surfaces face-to-face for obtaining three test samples. 14 ACS Paragon Plus Environment

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Blocking in the test samples was provoked under 12.45 kPa of pressure at 60°C during 30 min. Blocking resistance rate was reported on the scale of 0-10 (minimum and maximum, respectively). Tensile tests specimens were prepared following ASTM D882, where film specimens were cut with dumbbell shape of length 9.53 mm and cross section 3.18mm×1mm. Test was carried out according to ASTM D638 with an elongation rate of 25 mm/min. Film hardness was determined as the force measured in compression when the sample was penetrated 1mm with a 2mm cylinder plane-probe. Tensile and hardness test were carried out in a universal testing machine (INSTRON 3344), at 23 °C and 50% of RH. For each sample, the average values were reported from at least five tested specimens. Static contact angle (CA) of water on film surfaces was measured. To this effect, 120µm films were cast on a glass plate and dried at room temperature overnight. Then, distilled water droplets of 20 µL were deposited onto films surface. CA were obtained by the LBADSA method implemented in an ImageJ software.28 For each sample, the average CA was reported from ten measurements. 15 ACS Paragon Plus Environment


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Water and organic solvent resistance test were carried out onto film specimens of 1 mm of thickness and 10 mm in diameter. Film specimens were immersed in the tested solvent (water or MEK) at room temperature during 8 hours. Along test, specimens were withdrawn from the solvent and weighted after removing the excess of solvent from the the film surface with a paper absorbent, and immediately immersed again. Water and MEK absorption (AW and AMEK respectively) as the percentage of the solvent mass absorbed with respect to the dried film. In addition, the initial absorption rate was also calculated from the swelling curve and it was expressed as the percentage of water or MEK absorbed per minute (Awater/min and AMEK/min, respectively). Biodegradation ability of the hybrid films in composting condition was investigated. Film specimens (10 mm of diameter and 1 mm of thickness) were buried in a moisturized commercial compost with the following characteristics: total dry solid = 45% of the wet solids; non volatile-solids content = 40% of the wet solids; pH 6.5. Burial tests were run at 30 °C and the compost moisture was controlled constant at 55%. After 7 and 14 days of burying samples were


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withdrawn, carefully cleaned and dried in oven at 70°C. The percentage of film mass (Wloss) due to biodegradation was reported. 2.

Results and discussion In the current work, we investigate the synthesis of hybrid latexes with 25% of

proteins by miniemulsion polymerization. Protein formulation (Table 1) involves casein, as the major bio-component, while a small fraction of zein, a water repelling protein, is also incorporated in order to improve the water resistance of hybrid films, keeping constant the acrylic/proteins ratio. 2.1.

Miniemulsion and latex characterization

The results of the investigated miniemulsion polymerizations are summarized in Table 2. Under identical miniemulsification conditions and after 3 h of polymerization time, the final monomer conversion was around 95% when KPS was used as initiator, while it was a bit lower when employing TBHP. In addition, the use of methacrylated casein in the casein-base latexes (A/CF) produced a lower final x with respect to those where native casein was the incorporated biomaterial (A/CN). Amine groups of casein were reduced after casein methacrylation, and



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consequently the efficiency of TBHP for initiating the polymerization also decreased, since it involves the redox reaction between the hydroperoxide and an amine group of the casein.10,13

Table 2. Final results for the synthesis of hybrid latexes. Experiment x (%) dd (nm) dp (nm) Np/Nd PGE (%) IF (%) A

94

189

130

3.1

-

3

A/CN

89

223

170

2.3

33

95

A/CF

86

178

177

1.1

40

90

A/CNZ

90

248

190

2.2

43

93

A/CFZ

95

209

168

1.9

75

91

As can be seen in Table 2, all hybrid latexes presented a final dp smaller than dd, resulting in Np/Nd ratios between 1 and 2.3. This result indicates the presence of secondary nucleation by micellar or homogeneous mechanism. In addition, final dp values for the hybrid nanoparticles were higher than that of pure acrylic nanoparticles, due to the incorporation of a natural protein a stabilizer instead of an emulsifier. Also, an important secondary nucleation resulting Np/Nd higher than 3 was obtained in absence of proteins (A), probably due to the excess of emulsifier. Native casein presents an amphiphilic character with a critical micellar


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concentration of 0.1 mg/ml;6 however, the incorporation of methacrylic groups in functionalized casein affects its emulsifying capacity and promotes its polymerization (i.e., it acts as a surfmer).13 Likely, the polymerization capability of functionalized casein avoided its migration from polymer particles and therefore new polymer particles were formed by secondary nucleation, resulting in lower Np/Nd than when native casein was employed. An important point to consider in hybrid materials is the degree of compatibility between biomaterials and synthetic copolymers. Casein-based hybrid latexes synthesized with methacrylated casein (A/CF) showed higher PGE values than those obtained when native casein (A/CN) was incorporated. The previous methacrylation of casein with GMA significantly increased the PGE value, indicating an improvement in the degree of compatibility of acrylic/casein particles. It is in agreement with results by Picchio et al.13 However, A/CN showed higher PGE values than that reported by Picchio et al. (~20%) when native casein was used.13 This difference could be due to both: i)the employment of a different polymerization process (Picchio et al.13 used emulsion polymerization, instead of miniemulsion polymerization); and ii) GMA



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was added into the organic phase during the preparation of the miniemulsion, favoring the casein modification and increasing the PGE value. Also, it can be observed that when zein was incorporated (A/CNZ and A/CFZ) the PGE values were higher than their homologous latexes without zein. It was previously observed by Picchio et al,13 that PGE increases by reducing casein content. But in the current cases, the reduction of casein content was not significant for producing the observed PGE increases. These increase in PGE could be probably due to the high interactions reported between both proteins that hinders casein desorption from polymer particles. It has been reported that casein is an ideal stabilizer for zein particles due to absorption of amphiphilic protein molecules (casein), onto zein domains by hydrophobic interations.4,29 On other hand, all hybrid samples presented IF higher than 90%, independently of their protein composition. Considering that the IF of the hybrid materials is composed by the acrylic gel, the free protein and the acrylic-graftprotein copolymer; a high IF is indicating an important contribution of the content of grafted acrylic-protein due to IF attributed to pure acrylic (IF = 3 %) is very low. 20 ACS Paragon Plus Environment

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a)

b)

0.2 µm

0.2 µm

Figure 1. Particle morpholgy of hybrid latexes A/CF (a) and A/CFZ (b) obtained by TEM. Figure 1 shows particle morphology of nanoparticles obtained from latexes A/CF and A/CFZ, prepared at the same dilution. Sample synthesized only with casein as biomaterial (A/CF, Figure1a) presented compatibilized particles with smooth surfaces. However, when zein was incorporated (A/CFZ, Figure1b), particles presented a rougher surface. It is important to remark that the presence

of

separated

agglomerates

of

protein

was

not

observed,

demonstrating that miniemulsion polymerization allowed the synthesis of compatibilized acrylic/protein particles. In addition, TEM images obtained at low magnifications (20000X) evidenced a wide distribution of the particle size for both latexes, A/CF and A/CFZ (Figure S2 of SI). A broad particle size distribution 21 ACS Paragon Plus Environment


Page 22 of 57

was also obtained by DLS (Figure S3 of SI), with the presence of small particles, probably formed by secondary nucleation. 2.2.

Hybrid films formation

An important property of a polymeric dispersion is its ability to form smooth films. This property is characterized by the MFFT that defines the temperature range of application at which a latex uniformly coalesce. As it can be observed in Figure 2, all hybrid latexes produced clear films at room temperature with a MFFT below 2 °C. As it is known, a highly transparent film indicates the absence of big segregated phases in the films.30 In addition, the incorporation of zein in the formulation of the hybrid latex, turn the films a bit yellowish because this is the characteristic color of that protein.

Figure 2. Picture of films (with a hollow disc form) obtained from different formulations. In order to compare with the hybrid films properties, the physical mixture of pure acrylic latex (A) with casein and zein (PMix) was prepared, by keeping the 22 ACS Paragon Plus Environment

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same ratio acrylic/protein of hybrid latexes A/CNZ and A/CFZ(see “Physical mixture acrylic/proteins” section of SI.) In contrast with hybrid films, PMix film was opaque, heterogeneous and presented big segregates of zein (Figure S4 of SI). This heterogeneity is a consequence of the hydrophobic character of zein, which makes difficult its incorporation, and that most of the proteins remained uncompatibilized (evidenced by the very low value of PGE, Table S1). In hybrid films, it is essential to know their morphology for adequately understanding films performance. For this reason, morphology studies were carried out onto air-contact interface and cross-section cuts of films. Figure 3 shows AFM phase images for the protein-free film (A), and for two hybrid films containing methacrylated casein (A/CF and A/CFZ). For space reasons, only phase images are showed (at same phase scale) due to they provide image contrast between the soft (dark contrast) and hard phase (bright contrast). The surface and cross-section of pure acrylic film (Figure 3a-b) showed the formation of a homogeneous and continuous film, with a unique soft phase due to the complete coalescence of acrylic particles. In contrast, hybrid films (Figure



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3c-f), showed percolated films with a coalesced hard phase (protein phase) with small soft domains (acrylic phase).

212.10 Hz

400nm

400nm

400nm -352.38 Hz 101.43 Hz

400nm

400nm

400nm -129.44 Hz

Figure 3.AFM phase images (2000 nm×2000 nm) of pure acrylic film (a,b), A/CFfilm (c,d) and A/CFZ film (e,f).

It should be noted that the AFM technique does not allow to differentiate the proteins (casein and zein), due to both proteins have similar glass transition temperatures (~170 °C) and, therefore, similar hardness. Despite their high Tg, the film morphology showed that hybrid acrylic/protein particles were completely coalesced, mainly by the shell casein phase. Casein, which stabilize particles, is


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located in the particles shell, and therefore this protein favors film formation (i.e., particles coalescence) due to water acts as a plasticizer.16,31 It can be observed that, for both films (with and without zein) the protein phase was uniformly distributed surrounding the soft acrylic phase. Also, phase aggregation was not distinguished throughout the films, indicating that components achieved a high compatibilization and a restricted mobility of protein during film formation.12,13,32This observation is in agreement with the high compatibility indicated from the high PGE values (Table2).In contrast, AFM images for film surface obtained from the physical mixture PMix presented a continuous hard phase with the presence of big and irregular aggregates of acrylic particles (Figure S5 of SI), due to the lack of compatibilization between proteins and the acrylic polymer, as it was showed in Table S1 of SI. 2.3.

Film thermal properties

Table 3 summarizes the results of Tg and Td,max for hybrid films as well as for pure proteins and acrylic films. Table 3. Results of Tg and Td,max. Experiment

Tg (°C)

Td,max(°C)



A

-35.03

385.9

Casein

183.0 12

351.8

Zein

154.1 26

356.8

A/CN

-36.29

386.5

A/CF

-35.34

387.8

A/CNZ

-37.43

381.9

A/CFZ

-33.82

382.5

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All hybrid polymers presented a similar Tg, around -35 °C, coinciding with A, which has the same acrylic monomer composition (BA/MMA = 80/20). However, at least two Tg should be evidenced for these materials, one corresponding to the acrylic polymer and the other to the protein phase.12 Since, glass transition coincide with the beginning of their thermal decomposition for both proteins (~170 °C),26,27 hybrid films were analyzed by DSC measurements until 130 °C, and therefore higher Tg values were not reported. Native proteins presented a wide decomposition range with a Td,max of around 350 °C (Figure 4), and the pure acrylics film (A) exhibited a single weight loss step with a higher Td,max (386 °C). On the other hand, hybrid films showed a Td,max of around 386 °C, without significant variation for the A sample (Figure4). Similar results were previously reported for acrylic/casein films, where a high 26 ACS Paragon Plus Environment

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protein grafting extent did not significantly affect the nanocomposites degradation temperature.7In addition, the initial decomposition temperature (Td,i) for the hybrid films was lower than that for the pure acrylic film. This is in agreement to the Td,i of native casein and zein, which is consistent with the previous reports.26,33

Figure4.Thermogravimetric analysis for pure components and hybrid films.

2.4.

Mechanical behavior

Blocking resistance is a key parameter of polymer films that not stick or adhere to itself upon contact. Figure 5 shows average anti-blocking rate for pure acrylic and hybrid films (results of triplicate blocking measurements for each sample are summarized in Table S2 of SI). Acrylic film (A) had poor anti-



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blocking capability, with a total adherence between surfaces. In contrast, hybrid films showed major blocking resistance, due to the incorporation of hard materials (as proteins with high Tg) to a soft acrylic formulation improving blocking resistance, by reducing the tack adhesion energy and increasing the bulk storage modulus.1,12,34 Note that, when modified casein was employed (A/CF and A/CFZ) higher values of blocking resistance were obtained with respect to those with native casein. A/CFZ resulted in a completely non blocking film (the maximum anti-blocking grade) without traces of surface sticking.

Figure 5. Blocking resistance of pure acrylic and hybrid films.

Mechanical test results of hybrid films and pure acrylic film are showed in Table 4. As it is known, pure proteins films are highly brittle and show very little elongation; in contrast pure acrylic film, which is easily elongated (extension at 28 ACS Paragon Plus Environment

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break close to 3400%) at low tensile strength and presents a low hardness (Figure6). Pure BA/MMA copolymer presents a glass transition temperature (Table 3) below the test temperature (23 °C), and therefore is in its rubber-like state. As consequence, film A presents high deformations without elastic recovery (Figure 6).



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Table 4. Mechanical performance of acrylic/protein hybrid films. Comparison with an acrylic film.

Experiment

Tensile strength Elongation at break

Hardness

(MPa)

(%)

(N)

A

0.2±0.01

3395.7±135.82

1.0 ±0.04

A/CN

6.9 ±0.49

561.0 ±10.46

40.5 ±1.57

A/CF

8.6 ±0.15

387.8 ±24.93

44.7±1.58

A/CNZ

6.6 ±0.32

487.6 ±12.36

39.3 ±3.83

A/CFZ

6.8 ±0.18

305.8 ±10.05

50.8 ±1.64

Figure 6.Tensile test results for hybrid acrylic/protein films and pure acrylic film.

The incorporation of proteins, as a hard component, became the hybrid films less resistant to deformation than pure acrylic films, however they supported important deformations (> 300 %) and presented increased hardness (> 39 N). 30 ACS Paragon Plus Environment

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These results are in agreement with those reported elsewhere, where the incorporation of a hard protein phase improved mechanical properties.11,35 Furthermore, when employing the functionalized casein(A/CF and A/CFZ), materials were strengthened, decreasing their elongation at break but increasing tensile strength and hardness, compared to those obtained with native casein (A/CN and A/CNZ, respectively). Based on the PGE results, higher cross-linked films were obtained when methacrylated casein was used, which contributed to produce a cross-linked network, thus affecting mechanical performance. In addition, the zein content of hybrid films did not significantly affect their tensile behavior with respect to zein-free hybrid films. It was because, all the hybrid films had the same total protein content and both proteins have similar Tg. Despite that, an apparently small degradation of tensile strength and elongation capability was produced by the presence of zein. Note that when proteins were incorporated by simple physical mixture with the acrylic latex (PMix), the obtained film presented lower tensile strength and elongation ability than homologue hybrids (Figure S6 of SI), due to the lack of compatibility between phases.



2.5.

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Solvents resistance

Solvent resistance is an essential performance requirement for most film forming latex applications. While pure acrylic films have a high water resistance (8.5 % of water absorption after 8 hours of immersion), their resistant to organic solvents is poor. For this reason, the films from acrylic/protein nanocomposites were evaluated by measuring the static contact angle (CA) and the solvent resistance by immersion in water and MEK (as organic solvent). Contact angle measurements are widely employed to evaluate film surface hydrophobicity.36 Figure 7a summarizes the values of CA measured onto film surfaces of pure acrylic and hybrid acrylic/protein films. Hybrid casein-based films (A/CN and A/CF) showed lower CA than pure acrylic film, due to their high concentration of casein (25 % wbm), a protein of hydrophilic nature. Note that both films, with native (A/CN) and modified casein (A/CF) showed similar CA values, due to both latexes had the same casein content and similar PGE.


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a)

b)

A/CF

A/CFZ

Figure 7. Static contact angles of water on the pure acrylic and hybrid films surfaces (a); Picture of a water droplet on surfaces of hybrid films with and without zein (b).

The incorporation of zein (A/CNZ and A/CFZ) improved CA, by increasing their surface hydrophobicity compared with that of casein-based films (Figure 7b). The water repelling nature of zein counteracted hydrophilicity of casein, reaching a similar CA than that obtained in the pure acrylic film (~ 82°).Finally, it is worth noting that the proposed synthesis strategy was able to obtain hybrid films with a high fraction of renewable components into the global film formulation and with a surface hydrophobicity similar to that of pure acrylic film by replacing a small fraction of casein (the hydrophilic protein) by zein (the hydrophobic protein).



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Figure 8 shows the solvent absorption during 8 hours of film immersion in distilled water and MEK (AW and AMEK, respectively) at room temperature. The content of ionically-charged components of latexes, which remains trapped in interstitial areas of films, become a driving force for the migration of water into these interstitial areas of films and therefore increasing AW. An important drawback of hybrid casein-based films is their water susceptibility, because of their high content of casein, which is a highly hydrophilic macromolecule. Therefore, ungrafted casein domains presented in the hybrid films could be swollen with water. In this scenario, AW must depend on the grade of protein grafting, which modifies the hydrophilic character of casein,7 and on the presence of a compatibilized hydrophobic component, as it could be the zein. Figure 8a shows that water resistance of hybrid films was improved when the degree of grafting increased, resulting lower AW values for higher PGE. In addition, hybrid casein-based films containing zein (A/CNZ and A/CFZ) showed improved water resistance (AW final values equal to 232% and 117%, respectively), with respect to those materials containing casein as the only


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biomaterial (final AW values for A/CN and A/CF equal to312% and 282%, respectively).

Figure 8.Water (a) and organic solvent (b) absorption for pure acrylic and the acrylic-proteins films.

Results obtained for the A/CNZ film, which present similar values of PGE than both zein free casein-based films (A/CN and A/CF), demonstrated that water resistance could be notably improved by just substituting 5 % of the casein content by zein. In the case where the highest PGE was reached and zein was incorporated, A/CFZ film, the highest improvement on water absorption resistance was achieved. The solvent resistance results are shown in Figure 8b. The incorporation of natural proteins in acrylic latexes considerably improved the resistance to 35 ACS Paragon Plus Environment


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solvents. Thus, while film A was completely dissolved after a few minutes of immersion in MEK, hybrid films resisted the test without disintegrating. It is an evidence of the synergetic effect between both components, where the presence of proteins supplies a sufficient barrier to organic solvent.12 In this case it is observed that increasing PGE improved solvent resistance (lowering steady state solvent absorption). Also, in the case of film samples with similar PGE (A/CF, and A/CNZ) the incorporation of a hydrophobic protein as zein in A/CNZ degrades solvent resistance.

Figure 9. Initial solvent absorption rate in water (blue) and organic solvent (red) for hybrid films

The analysis of the initial absorption rate of the films in both solvents gives some information about films behavior after their solvent exposition. Figure 9 36 ACS Paragon Plus Environment

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shows the initial absorption rate of hybrid films, in water and MEK. For most hybrid films, it can further be observed that, the absorption rate is higher in water than in MEK, which is in agreement with the steady state values observed for both solvents. Also, hybrid films showed that initial water absorption rate was reduced by increasing PGE (Table 2) and by incorporating zein. In contrast, the film obtained from the simple physical mixture of pure acrylic latex with both proteins (PMix) did not show a synergy between the hydrophilic and hydrophobic characters of the combined materials. Figure S7 of SI demonstrated that PMix film did not resist the immersion in solvents (water and MEK), presenting a great loss of mass during the test, due to the low compatibility reached between proteins and acrylic polymer. Previous results indicate that the use of native zein combined with the methacrylated casein for latexes synthesis by miniemulsion polymerization, was an effective method for producing compatibilized hybrid acrylic/proteins materials with high content of renewable components and with a high potential for industrial coating applications.



2.6.

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Soil biodegradation

Soil biodegradation results for pure acrylic films (A) and methacrylated caseinbased films (with and without zein) are presented in Figure 10, as the weight loss of films after 7 and 14 days into organic compost. Hybrid films lost almost 15% of their initial weight, because natural proteins tend to hydrolyze and degrade under composting condition. Despite of the acrylic phase was not degraded, the incorporation of natural proteins improved film biodegradability.

Figure 10. Hybrid films biodegradation after burying 7 and 14 days into organic compost.

For A/CFZ film, the degree of decomposition was a bit lower than for caseinbased films. This behavior was rather expected because zein possesses water repelling properties, and therefore zein‐based materials are more resilient to the action of microorganisms.37 38 ACS Paragon Plus Environment

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2. Conclusions Waterborne hybrid nanoparticles containing two proteins (casein and zein) as biopolymer, were synthesized by emulsifier-free miniemulsion polymerization. This polymerization method proved to be an adequate process for incorporating zein (a hydrophobic protein) into highly compatibilized hybrid nanoparticles. In contrast, the product obtained by physical mixing an acrylic latex with both proteins presented phases separation in both dispersion and film, due to the lack of compatibility between the components. Hybrid films obtained from casein-based latexes showed enhanced mechanical properties (with a maximum elongation of almost 500%), antiblocking properties and soil biodegradation (nearby 15%), keeping low MFFT and similar Td,max, with respect to the homologous pure acrylic film. Compared with native casein, the incorporation of previously methacrylated casein improved

the

compatibilization

of

acrylic-proteins

nanoparticles

and

consequently their final properties. However, an important drawback of caseinbased films is their water susceptibility (contact angle close to 75°), due to the hydrophilic character of this protein. The incorporation of a small fraction of zein



Page 40 of 57

significantly improved the water resistance of hybrid films, and increased their surface hydrophobicity (contact angle equal to 83°), without significantly degrading their mechanical and thermal properties. These hybrid films are attractive materials with a high potentiality for industrial applications as coatings. Acknowledgements The financial support received from CONICET, UNL, and ANPCyT(all of Argentina) is gratefully acknowledged. We also acknowledge to the Physics of Surfaces and Interfaces Laboratory (IFIS Litoral, Santa Fe, Argentina) for the use of their SPM equipment. References (1)

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AbstractGraphic



Figure 1. TEM images of the hybrid latexes A/CF (a) and A/CFZ (b) 211x125mm (96 x 96 DPI)


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Figure 2. Picture of films (with a hollow disc form) obtained from different formulations. 211x41mm (96 x 96 DPI)



Figure 3. AFM phase images (2000 nm ×2000 nm) of pure acrylic film (a,b), A/CF film (c,d) and A/CFZ film (e,f). 152x101mm (96 x 96 DPI)


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Figure 4. Thermogravimetric analysis for pure components and hybrid films. 151x123mm (300 x 300 DPI)



Figure 5. Blocking resistance of pure acrylic and hybrid films. 150x113mm (300 x 300 DPI)


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Figure 6. Tensile test results for hybrid acrylic/protein films and pure acrylic film. 153x124mm (300 x 300 DPI)



Figure 7. Static contact angles of water on the pure acrylic and hybrid films surfaces (a); Picture of a water droplet on surfaces of hybrid films with and without zein (b). 152x56mm (96 x 96 DPI)


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Figure 8. Water (a) and organic solvent (b) absorption for pure acrylic and the hybrid films. 216x92mm (300 x 300 DPI)



Figure 9. Initial solvent absorption rate in water (blue) and organic solvent (red) for hybrid films 149x112mm (300 x 300 DPI)


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Figure 10. Hybrid films biodegradation after burying 7 and 14 days into organic compost. 152x112mm (300 x 300 DPI)


proteins nanocomposites with enhanced

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