Amyloid-Like Protein Coating as a Barrier Against Plasticizer Leaching

Open AccessCCS ChemistryRESEARCH ARTICLES12 Jun 2024Amyloid-Like Protein Coating as a Barrier Against Plasticizer Leaching Facui Yang†, Mengjie Li†, Jingwen Xiong, Xiaopeng Lv, Xuehao Ma, Weixing Chen and Peng Yang Facui Yang† Shaanxi Key Laboratory of Photoelectric Functional Materials and Devices, Engineering Research Center of Light Stabilizers for Polymer Materials, Universities of Shaanxi Province, School of Materials and Chemical Engineering, Xi'an Technological University, Xi'an 710021 , Mengjie Li† Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710119 , Jingwen Xiong Shaanxi Key Laboratory of Photoelectric Functional Materials and Devices, Engineering Research Center of Light Stabilizers for Polymer Materials, Universities of Shaanxi Province, School of Materials and Chemical Engineering, Xi'an Technological University, Xi'an 710021 , Xiaopeng Lv Shaanxi Key Laboratory of Photoelectric Functional Materials and Devices, Engineering Research Center of Light Stabilizers for Polymer Materials, Universities of Shaanxi Province, School of Materials and Chemical Engineering, Xi'an Technological University, Xi'an 710021 , Xuehao Ma Shaanxi Key Laboratory of Photoelectric Functional Materials and Devices, Engineering Research Center of Light Stabilizers for Polymer Materials, Universities of Shaanxi Province, School of Materials and Chemical Engineering, Xi'an Technological University, Xi'an 710021 , Weixing Chen *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Shaanxi Key Laboratory of Photoelectric Functional Materials and Devices, Engineering Research Center of Light Stabilizers for Polymer Materials, Universities of Shaanxi Province, School of Materials and Chemical Engineering, Xi'an Technological University, Xi'an 710021 and Peng Yang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710119 https://doi.org/10.31635/ccschem.024.202404046 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Phthalate plasticizers can cause reproductive system problems and women's infertility as an endocrine interferon due to easy migration or leaching from plastics. Most of the coatings used for plastic surface modification exhibit limited efficacy in preventing plasticizer migration, and have the disadvantages of complex preparation methods and lack of interfacial biocompatibility. Herein, a dense proteinaceous/polysaccharide coating self-assembled from amyloid-like aggregates is formed on the surface of plastic. The coating exhibits excellent antiplasticizer migration properties and stable interfacial adhesion robustness, thereby guaranteeing optimal effectiveness when subjected to external forces such as bending, stretching, tearing, and flowing water, in order to comply with the migration limit set by the European Union Regulation. The leaching amount of plasticizer is reduced 83.8–99.9% in different leaching media compared to the uncoated plastics. Additionally, for medical plastics with coated phase-transitioned lysozyme/sodium alginate, including hemodialysis tubes, infusion tubes, and blood bags, the plasticizer concentration in the leaching solution was reduced by ∼92%. The coating also exhibits an excellent resistance to the nonspecific adsorption of active substances (drugs, platelets, and bacteria). This study provides a strategy for application of biopolymer-based coatings in food and medical packaging materials toward the inhibition of plasticizer migration. Download figure Download PowerPoint Introduction A phthalate is a small molecule and does not chemically bond with a plastic matrix, and it can easily leach out and migrate into the contacting biological environment, posing a threat to both nature and organisms.1–3 Lost economic productivity is approximately $40 billion/year due to phthalate exposure, which lead to 91,000 to 107,000 premature deaths per year between the ages of 55 and 64. Nonetheless, phthalates remain the predominant plasticizer, with a production capacity surpassing 8.4 million tons, accounting for approximately 85% of all consumed plasticizers.4,5 Among phthalates, di-(2-ethylhexyl) phthalate (DEHP) is the most widely used, accounting for more than 50% of the phthalate production. DEHP is mainly used in polyvinyl chloride (PVC) plastics, estimated to reach 60 million tons globally by 2025, ranking behind polyethylene (PE) and polypropylene (PP).6 Approximately 33% of plastic-based disposable medical products are fabricated using a flexible PVC plasticized with DEHP. The DEHP content in the flexible PVC is usually up to 30–50 wt %.7 As early as 2008, DEHP was categorized as a CMR 1B (carcinogenic, mutagenic, and reprotoxic substances) by the European Union because of its negative effects of disrupting endocrine systems.a However, in medical devices, DEHP is still in use, even for the treatment of vulnerable individuals such as pregnant women, neonates, and infants in general.8–10 Furthermore, plastics [e.g., polyethylene terephthalate (PET)] used to package food and drinks also suffer from plasticizer migration, particularly in numerous developing countries where recycling of plastic bottles to store drinking water and food is common.11,12 Therefore, a provision of the effective inhibition of the migration of plasticizers in plastics is an important global challenge. Several strategies are used to inhibit the migration of plasticizers, including the use of high-molecular weight novel plasticizers, addition of nanoparticles in the plastic matrix, and modification of the surface of the plastic by physical (e.g., plasma treatment, ultraviolet irradiation, radiation irradiation) or chemical (e.g., surface modification including cross linking, grafting, and coating) methods.13,14 Nevertheless, novel plasticizers exhibit drawbacks such as exorbitant costs and reduced plasticization efficacy, particularly under low temperature conditions. Furthermore, these plasticizers will still migrate from plastic films with longer times. Among these methods, the surface modification does not have a large impact on the main structures of plastic materials. A specific surface structure that can inhibit the migration of plasticizers can be obtained by modification. Nonetheless, neither physical nor chemical surface modification methods can fully inhibit plasticizer migration. For example, surface cross linking causes a color change of products, reduces the flexibility, and weakens the mechanical properties, while surface grafting suffers from technically sophisticated problems. The most studied surface coating is an oxide barrier coating (e.g., silicon oxide layer),15–17 which is still technically complex, expensive, and prone to fragmentation and peeling from the plastic particularly upon deformation of flexible plastics.15 Furthermore, the surfaces of medical-grade plastic products require consideration of the adsorption and penetration of drugs on the coating, which may affect the stability of the active ingredient of the drug or hinder the control of the concentration of the delivered drug.18–20 Considering the above concerns, it is necessary to develop a coating that can effectively inhibit the migration of the plasticizer, and has the advantages of low cost, simple preparation technology, good biocompatibility, and reliable adhesion robustness, with a negligible influence on active substances encapsulated in plastic containers. In this study, we utilize phase-transitioned lysozyme (PTL) through the reduction of intramolecular disulfide bonds by tri(2-carboxylethyl)phosphine (TCEP) to assemble an amyloid-like protein coating on the surface of plastics via a simple dip coating at room temperature.21–23 With effective incorporation of sodium alginate (SA), the resultant PTL/SA coating with stable adhesion properties could serve as a modified coating against plasticizer migration, while keeping excellent resistance to the nonspecific adsorption of active substances (drugs, platelets, and bacteria) (Scheme 1). The coating largely reduces (83.8–99.9%) the leaching of DEHP in different leaching media (n-hexane, 95% ethanol, 50% ethanol, and 3% acetic acid) compared to uncoated plastics and exhibits sufficient stability in the leaching solution to meet the European Union Regulation (EUR) migration limit. By keeping good biocompatibility, such a biobarrier may not only reduce the harm of plasticizers in food packaging to the human body, and but can also potentially be a very attractive replacement for some conventional medical coatings. Scheme 1 | Schematic of the PTL/SA coating inhibiting the migration of plasticizer with good biocompatibility. Download figure Download PowerPoint Experimental Methods The preparation of the amyloid-based coating The plastic films were immersed in a phase transition solution of lysozyme and TCEP. The phase transition solution of lysozyme was freshly prepared by mixing lysozyme with TCEP. This method of preparation and detailed coating procedure on a substrate was carried out as described by our previous papers.21 The cleaned plastic substrates were immersed into a lysozyme phase transition solution containing equivoluminal of 20 mg/mL lysozyme and 50 mM TCEP with pH 7.0 (adjust the pH by 5 M NaOH). The substrate was then incubated at room temperature for 2 h. Then, a PTL coating was formed onto the surface of substrates. By this method, the PTL coating was stably adhered on the surface of substrates and was rinsed with ultrapure water to remove the phase transition mixture on it. After that, PTL-coated plastics were immersed into 2 mg/mL SA solution and incubated at room temperature for 2 h, then rinsed with ultrapure water and dried by nitrogen. The migration test of plasticizer We adopted acidic aqueous solution (3% acetic acid), ethanol (50%, 95% ethanol), aliphatic hydrocarbons (n-hexane and isooctane) to simulate aqueous food, alcohol food, and fatty food, respectively. The barrier effect of the PTL/SA coating on the migration of dioctyl phthalate (DOP) and DEHP was assessed by UV–vis spectroscopy. For the DOP and DEHP migration test, the samples of PTL/SA-coated and uncoated plastic film were completely immersed in 2 mL of n-hexane solution. The system was maintained at room temperature for 1 h and the amount of plasticizer leached by plastic film samples was measured by UV–vis. A UV–vis spectrophotometer was used to obtain the UV–vis absorption spectrum. The absorption values at a 275 nm wavelength were compared between coated and uncoated plastic film. The leaching ratio (RL) is defined as the ratio of the plasticizer concentration (Ct) in coated plastics to that in uncoated plastics (C0) in the leaching solution. The leaching ratio (RL) and the barrier ratio (Rb) are calculated using eqs 1 and 2, respectively. R L = C t C 0 × 100 % (1) R b = C 0 − C t C 0 × 100 % (2) The migration amount of plasticizer was calculated according to the following eq (3): q t = C t · V 1000 · S (3)wherein, qt, migration amount, mg/cm2; Ct, concentration of plasticizer in the detected stimulant solution, mg/L; V, volume of stimulant solution, mL; and S, contact area between the sample and stimulant solution, cm2. For the medical plastic, 10 cm of the inner wall of dialysis tube and infusion tube and 450 mL of the inner wall of blood bag were coated with PTL/SA coating. Then, the PTL/SA-coated and uncoated tubes were filled with 95% ethanol and incubated at 37 °C for 1 h. The amount of plasticizer leached by medical plastic samples was measured by gas chromatography–mass spectrometry. Sample treatment before testing: first evaporate ethanol completely and then add 2 mL n-hexane to dissolve the plasticizer, and dilute the sample 100 times before sample injection. The GC column was programmed as follows: the temperature was initially held at 100 °C for 1 min, thereafter heated at a constant rate of 20 °C/min up to 250 °C, then held at 250 °C for 0.5 min, and finally heated at a constant rate of 15 °C/min up to 280 °C for 4 min. Further details of the preparation of plastic film, materials characterization, biocompatibility experiment of coating, and adhesion stability test of coating may be found in the Supporting Information. Results and Discussion Preparation and characterization of an amyloid-like protein coating When lysozyme was dissolved in water with the addition of TCEP at pH 7.0, a rapid lysozyme conformation change occurred after its disulfide bonds were broken down by TCEP, inducing the assembly of amyloid-like aggregates to form a PTL coating.24,25 The positive charge on the surface of the PTL coating can adsorb the linear-molecule polyanionic polysaccharide SA to form a PTL/SA complex coating (Figure 1a). The thickness of the PTL/SA coating was ∼210 nm. The surface of the PTL coating contains proteinaceous microparticles aggregated from oligomers,26 which appear on the surface of the coating in a semiembedded state (Figure 1b). After the coating of such PTL/SA layer on the surface of the PVC film, the coating has a typical protrusion structure derived from close-packed amyloid-like aggregates (Figure 1c and Supporting Information Figure S1). The C peaks in the X-ray photoelectron spectrum are considerably broadened (Figure 1d and Supporting Information Figure S2), and sodium peaks for SA appear (Figure 1e). In addition, the attenuated total reflectance Fourier-transform infrared spectrum shows that a C–O–C vibration absorption peak appears at 1100 cm−1 after SA is adsorbed ( Supporting Information Figure S3). As further elucidated by laser scanning confocal microscopy (LSCM), the use of a fluorescein isothiocyanate-conjugated SA (SA-FITC) indicates that the SA has bound to the surface of the PTL membrane (Figure 1f). The PTL/SA coating was transparent in the wet state (Figure 1g). The optical transmittance under visible light was 80–90% (Figure 1h). This property of the coating has important implications for its use in liquid environments without affecting the transparency of the plastic substrates. Before the coating, the surface wettabilities on various plastic substrates (PE, PET, polycarbonate (PC), and PP) are different. The water contact angle of each substrate surface was consistently 72° after the modification by the PTL coating ( Supporting Information Figure S4), which indicates a successful surface modification on plastic surfaces. When SA was combined with the PTL coating, the surface of PTL/SA became more hydrophilic, and the corresponding water contact angle was further decreased to 23° (Figure 1i), which is beneficial to enhance the antifouling behavior of the coating surface. In addition, it can be seen that the elastic modulus and tensile strength of the PE film increased in the presence of coatings ( Supporting Information Figure S5). This improvement in mechanical properties can be attributed to the stable adhesion of densely packed amyloid-like aggregates to the surface of the plastic film and the formation of a uniform coating. Therefore, the PTL/SA coating can enhance the mechanical properties of plastics, and does not affect the use of the film. Figure 1 | (a) Schematic of the preparation of the PTL/SA coating. (b) Representative SEM images of a cross section (left) and surface (right) of PTL/SA. (c) Atomic force microscopy images of the PVC (left) and PTL/SA-coated on the surface of PVC (right); scale bars, 1 μm. (d, e) High-resolution C1s and Na1s spectra of the PTL/SA coating. The deconvolution of the C1s peak indicates that the coating surface had multiple functional groups, including mainly aliphatic carbon (C–H/C–C), amines (C–N), hydroxyls (C–O), amides (O=C–N), and carboxyl groups (O=C–O). (f) LSCM images of the PTL/SA-FITC-coated PVC. (g) Photographic images to show PTL/SA-coated on glass and PVC in the dry and wet states. (h) Transmittance of the PTL/SA-coated on a quartz glass in the dry and wet states. (i) Water contact angles of the PVC and PTL/SA-coated PVC substrates. Download figure Download PowerPoint Leaching evaluation of the plasticizer Plasticizer leaching typically occurs by diffusion through internal voids in bulk materials. In the case of the present PTL/SA coating, internal voids are formed by the dense packing of protein oligomer nanoparticles. The void size could be conveniently adjusted by the protein concentration and composite additives.24,25 In this regard, the pore size of the PTL membrane was determined to be ∼2 nm when the concentration of lysozyme was 20 mg/mL (Figure 2a). After the introduction of SA into the PTL, according to a preliminary analysis, the pore size of the PTL/SA membrane was reduced to 1.3 nm by the permeation of polyethylene glycol (PEG) molecules of different molecular weight (Figure 2a and Supporting Information Figure S6). The molecular configurations of DEHP and DOP are as ellipses, with molecular sizes of approximately 1.6 and 1.5 nm, respectively (Figure 2b). Therefore, the pore size of the PTL/SA membrane is smaller than the size of the plasticizer molecule. Thus, the coating is expected to prevent the leaching of the plasticizer from the plastic substrate (Figure 2b). Figure 2 | (a) Probability density function showing the pore size distribution of the PTL/SA membrane. (b) Molecular structures of DEHP and DOP. Schematic of the plasticizer migration after the surface modification of the plastic substrate with PTL/SA. (c) Schematic of the PTL/SA coating on PS for the test of plasticizer migration. Effects of PTL (d) or PTL/SA (e) coatings prepared at different lysozyme concentrations on the leaching of plasticizers, and schematics of plasticizer passage through the PTL or PTL/SA coatings (insets). (f) Influence of the combination of the PTL layer (the concentration of lysozyme is 20 mg/mL) with different concentrations of SA on the leaching of plasticizer, and leaching ratio of the plasticizer in n-hexane for different soaking times (inset). (g) Leaching amount of the plasticizer in different medical plastic products before and after surface modification by PTL/SA, after immersion in 95% ethanol for 1 h at 37 °C. (h, i) Plasticizer leaching amounts of different plastics before and after modification by PTL/SA, after immersion in 3% acetic acid and 50% ethanol at 37 °C for 10 days, respectively. Download figure Download PowerPoint To assess the effect of the amyloid-based coating on the inhibition of plasticizer leaching, DOP was added to a polystyrene (PS) toluene solution to cast a film according to the traditional method of blending into a film27 (for details, see Supporting Information experimental section). The surface of the PS film was then modified with amyloid-like aggregates to form a PTL/SA coating (Figure 2c). Considering the practical application, n-hexane was selected as a lipophilic substance in the migration test.28 As the concentration of lysozyme increased from 2 to 30 mg/mL, the leaching ratio (RL) of the PTL coating to the plasticizer decreased from 70% to 4.5% (Figure 2d and Supporting Information Figure S7a–c). As a response to the increased lysozyme concentration, the coating thickness of the PTL membrane increases, resulting in smaller pores.24 Additionally, the increase in lysozyme concentration makes the phase transition faster, and numerous microparticles are formed on the membrane surface and gradually become larger ( Supporting Information Figure S8). Such a result is more helpful to extend the time for plasticizer passage through the coating due to blockage of microparticles on the membrane surface. During the accelerated phase transition, the lysozyme reaches a concentration of 50 mg/mL, and thus the coating surface becomes rougher, and the leaching ratio is slightly increased by 16.4% (Figure 2d). To further decrease the leaching ratio of the plasticizer, the linear-molecule SA was introduced into the surface of the PTL coating by an electrostatic self-assembly to seal the gaps between amyloid aggregates. The PTL/SA coating has a better inhibitory effect on the plasticizer leaching. The leaching ratio was decreased to 2.2% when the lysozyme concentration was larger than 20 mg/mL (Figure 2e and Supporting Information Figure S7d). The plasticizer leaching ratio reached equilibrium when the SA concentration exceeded 1 mg/mL (Figure 2f and Supporting Information Figure S7e). The adsorption equilibrium time of the PTL coating on SA was also investigated. After adsorption of SA for 20 min, the plasticizer leaching ratio for the PTL/SA coating was reduced to 11.3%. The ratio was further reduced to 2.1% after the electrostatic self-assembly of SA for 2 h (Figure 2f and Supporting Information Figure S7f). According to the fluorescently labeled SA (SA-FITC), the SA density on the PTL coating was estimated to be 0.72 chain/nm2, providing a sufficient coverage on the PTL surface ( Supporting Information Figure S9). After the migration, unlike the whitened unmodified PS film, the PTL/SA-coated PS still had good light transparency ( Supporting Information Figure S10). In addition, at low mass of DOP the PS and DOP result in that hinder the of DOP ( Supporting Information Figure When the DOP mass ratio reaches up to DOP to increase = of DOP to of the plasticizer in the PS film with DOP = and the leaching ratio was reduced by compared with PS without PTL/SA coating. that the PTL/SA coating can effectively prevent the leaching of the DOP plasticizer from the PS on the and nature of the contact substance to be may be selected in of the corresponding of For example, blood is a lipophilic and 95% ethanol can be used as a contact for medical on the above we further used the PTL/SA coating to the inner of medical PVC products infusion and blood and then a DEHP plasticizer leaching test with 95% ethanol at 37 °C for 1 h. The plasticizer leaching of hemodialysis tubes, infusion tubes, and blood were reduced to and after the modification with the PTL/SA coating ( Supporting Information Figure respectively. The corresponding concentrations in the leaching were and obtained by gas chromatography–mass respectively (Figure The plasticizer concentrations in the contact were reduced compared to the uncoated plastics. In addition to medical PVC plastic products, plastic films used for food packaging PET, and The PTL/SA coating was then to the surfaces of these plastic films to the of According to the of food we selected different food including 3% acetic acid and 50% ethanol to study the leach of plasticizers DEHP from plastic films (e.g., PET, and The modified plastic films with the PTL/SA coating were in different food for 10 at °C according to the The migration amount of plasticizer was to the of plastic and content of the The leaching amount of the plasticizer in the plastic with the PTL/SA coating is reduced in 3% acetic acid compared to the uncoated while the leaching amount from is reduced approximately (Figure 50% ethanol is more than 3% acetic the migration amount is still reduced under the of the PTL/SA coating (Figure and Supporting Information Figure compared with the uncoated the plasticizer leaching were reduced to and after soaking in 3% acetic acid and ethanol at room temperature for 30 days, respectively ( Supporting Information Figure Therefore, the PTL/SA coating can still effectively reduce the migration amount of the plasticizer compared to the uncoated plastics, as most an ethanol content In addition, we compared PTL/SA coatings with and coatings for plastic surfaces. The that the barrier ratio of the PTL/SA coating for DEHP exceeded the barrier (Rb) of and coatings by and respectively ( Supporting Information Figure Furthermore, the of PTL/SA is more and better for food and medical packaging materials compared to traditional plastic coatings. of plasticizers under different The migration of plasticizer is by including the and stability of the coating. We the amount of plasticizer migration from PVC in 95% ethanol and at different times using the PTL/SA coating, respectively. The plasticizer migration of the PTL/SA coating a before equilibrium (Figure The migration rate of the plasticizer from the plastic to the can be using a − = where is the migration amount at time t is the specific migration at the equilibrium time is the migration rate constant and t is the migration time at contact by The that the migration rate = was reduced by the first 2 h due to the barrier properties of the PTL/SA compared to the uncoated PVC (Figure which to the plasticizer leaching into the water, food, and The plasticizer migration rate of the uncoated PVC film in was found to be than that in 95% ethanol (Figure and Supporting Information This that the migration rate of phthalate molecules in leaching media was than that in the plasticizer migration rate of the PTL/SA-coated PVC film in was to be than that in 95% ethanol This can be attributed to the of the PTL/SA which as a barrier layer preventing contact between the leaching and the surface of the plastic film, the rate of the leaching into the plastic film is Figure | (a) amount of plasticizer from PVC and into 95% ethanol and at different respectively. (b) migration of plasticizer from PVC and into 95% ethanol and (c) of plasticizer in water with different pH (d) of the PTL/SA-coated PVC under external forces and flowing (e) Plasticizer leaching amounts after is in 95% ethanol after for tensile of peeling times using the and with a of 100 (f) SEM images of the PTL/SA coating on PVC after 1 h of contact with 95% The coating was times before (g)