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Article A Double-Switch Temperature-Sensitive Controlled Release Antioxidant Film Embedded with Lyophilized Nanoliposomes Encapsulating Rosemary Essential Oils for Solid Food

Xi Chen 1 , Qing Long 1, Lei Zhu 2,*, Li-Xin Lu 1,3,*, Li-Nan 1, Liao Pan 1,3, Li-Jing Lu 1,3 and Wei-Rong Yao 4

1 Department of , Jiangnan University, Wuxi 214122, China; [email protected] (X.C.); [email protected] (Q.L.); [email protected] (L.-N.S.); [email protected] (L.P.); [email protected] (L.-J.L.) 2 China National Center for Food Safety Risk Assessment, Beijing 100022, China 3 Key Laboratory of Advanced Food Manufacturing Equipment and Technology of Jiangsu Province, Jiangnan University, Wuxi 214122, China 4 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; [email protected] * Correspondence: [email protected] (L.Z.); [email protected] (L.-X.L.)

 Received: 3 November 2019; Accepted: 29 November 2019; Published: 3 December 2019 

Abstract: In order to match the solid food oxidation during logistics and storage process under severe high temperature, a double-switch temperature-sensitive controlled release antioxidant film embedded with lyophilized nanoliposomes encapsulating rosemary essential oils (REOs) was prepared. The double switch temperature at 35.26 and 56.98 ◦C was achieved by development of a temperature sensitive polyurethane (TSPU) film. With biaxially oriented terephthalate (BOPET) as a barrier layer, the intelligent complex film was prepared via coating the TSPU embedded with lyophilized nanoliposomes encapsulating REOs on BOPET. The results indicate that the REO is well encapsulated in nanoliposomes with encapsulation efficiency (EE) of 67.3%, high stability and lasting antioxidant effect during 60 days. The incorporation of lyophilized nanoliposomes containing REOs into TSPU remains the double-switch temperature-sensitive characteristic of the prepared TSPU. In agreement with porosity and WVTR results, the diffusion coefficient (D) of the antioxidant complex film sharply increases respectively at two switching temperatures, indicating that the intelligent double-switch temperature-sensitive controlled release property is functioning. Furthermore, compared with films directly added with REO, the lower Ds of films added with lyophilized nanoliposomes encapsulating REOs provides a longer-lasting antioxidant activity. Thus, the acquired controlled release antioxidant film sensitive to temperature at 39.56 and 56.00 ◦C can be potentially applied for protection of solid food during distribution and storage process under severe high temperatures.

Keywords: temperature-sensitive; double-switch; controlled release; antioxidant; nanoliposome; rosemary essential oils

1. Introduction Protecting food from deterioration and flavor altering caused by oxidation has become one of the major challenges for the food industry [1]. Antioxidant packaging is an effective technology to delay the oxidation process by scavenging free radical and interdicting peroxide [2]. Moreover, antioxidant

Materials 2019, 12, 4011; doi:10.3390/ma12234011 www.mdpi.com/journal/materials Materials 2019, 12, 4011 2 of 15

film with controlled release function can provide a better antioxidant effect for the ability of adjusting the release rate of antioxidant to satisfy the demand of food to be oxidized [3]. However, most of the studies on controlled release antioxidant film focused only on the sustained release of antioxidant, in which the obtained films cannot stimulate the release rate of antioxidant according to the alteration of environmental conditions such as pH, temperature, and illumination [4]. While the oxidation rate is significantly affected by the logistics and storage environment, which may dramatically change during the distribution and storage process. Thus, it is necessary to exploit the controlled release of antioxidant films that are sensitive to environmental factors to further protect food from severe circumstances. The environmentally responsive smart membrane can respond quickly to external environmental stimulus and convert external signals into internal signals (internal structure, pore size, etc.,) to change the performance of the materials [5]. Among all the environmental factors, temperature is one of the most significant factors influencing the oxidation process. Therefore, the academic community has demonstrated an interest in the development of temperature responsive polymer and hydrogel [6,7]. Temperature sensitive polyurethane (TSPU) is one of the excellent temperature responsive materials and has a significant phase change in a certain temperature range [8]. The phase change will lead to a sudden change of free volume, and cause the permeability change [9,10]. This character of TSPU provides a possibility to control the release speed of active substance inside the film. Hence, Dong et al. [11] applied TSPU with a single switch temperature to control the release of directly incorporated carvacrol and cinnamyl aldehyde as antimicrobials, presenting superior protection on food compared with PU without temperature sensitive property. Nevertheless, to the author’s knowledge, there has been no research on TSPU based temperature-sensitive controlled release antioxidant film. In addition, the single switch TSPU provides a relatively narrow range of stimulating temperature which may limit the application in practice. Therefore, TSPU with multiple switch temperature needs to be developed for the temperature-sensitive controlled release of antioxidants. Among the most widely utilized antioxidant, rosemary essential oil (REO) is a typical natural antioxidant essential oil with perfect antioxidant effect and approval by FDA and European Commission [12]. Specifically, the main functional ingredients of REOs include carnosic acid, carnosol, rosmarinic acid, and rosmanol. Carnosic acid is the compound with the highest antioxidant activity in rosemary essential oil, about three times higher than carnosol and about seven times higher than synthetic antioxidants butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) [13]. The antioxidant activity of rosmarinic acid is stronger than that of vitamin E, and the antioxidant activity of rosmanol is about five times that of BHT and BHA. Besides, the volatile essential oils can diffuse into the space of package without contact with foods, providing a possible solution for the problem of oxidation of solid foods. Thus, REO was chosen as the antioxidant in this study. However, due to the instability and volatility of essential oils, directly blending REOs and TSPU may lead to a rapid release of antioxidant, thus nanoliposome encapsulation technique was introduced for its stable, non-toxic and sustained release properties [14]. Although the nanoliposomes encapsulating essential oils have been successfully embedded in chitosan [15,16] and protein [17] films, the incorporating of nanoliposomes containing REOs into TSPU has not been studied. Furthermore, the influence of nanoliposomes containing REOs on the structures, morphology and thermal characteristics of TSPU requires further study. The aim of this was to prepare a double-switch temperature-sensitive controlled release antioxidant film based on TSPU incorporated with lyophilized nanoliposomes containing REOs as temperature sensitive layer, and BOPET as barrier layer. Then investigate how the lyophilized nanoliposomes containing REOs influence structures, morphology and thermal characteristics of TSPU, and how the double-switch TSPU control the release of REO to realize a longer-lasting antioxidant protection for solid food. Materials 2019, 12, 4011 3 of 15

2. Materials and Methods

2.1. Materials

Polycaprolactone (PCL, Mw = 4000, CP) was purchased from Suyu raw materials business department (Dongguan, China). 4,40-diphenylmethane diisocyanate (MDI, 98%) was obtained from J&K Scientific Co., Ltd. (Beijing, China). Polyethylene glycol (PEG, Mw = 2000, CP), 1,40-butanediol (BDO, AR), N,N-dimethylformamide (DMF, AR), soy lecithin (PC, BR), cholesterol (CH, 0.995), ≥ Tween 80 (T-80, CP), polyvinylpyrrolidone (PVP, 0.995) and anhydrous ethanol (GR) were bought ≥ from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Polyurethane (PU) was obtained from Shunte Plastic Co., Ltd. (Guangdong, China). Biaxially oriented polyethylene terephthalate (BOPET) was purchased from Yiwei Mechanical and Electrical hardware Co., Ltd. (Shanghai, China). Rosemary essential oil (REO, RG) was received from Chroma Dex Corporation (USA); 2,2-diphenyl-1-picrylhydrazyl (DPPH, 97.0%, HPLC) was acquired from Aladdin Biotechnology Co., ≥ Ltd. (Shanghai, China).

2.2. Preparation of Lyophilized Nanoliposomes Containing REOs Nanoliposomes containing REOs were prepared by thin film hydration method [18]. Briefly, 1.00 g of REO, 6.00 g of soy lecithin and 1.00 g of cholesterol were fully dissolved in 150 mL of chloroform. Solvents were evaporated under reduced pressure at 30 ◦C using a rotary evaporator. The obtained lipid film on the inner wall was hydrated with 300 mL of phosphate buffer (pH = 6.8, 1.224 g of sodium dihydrogen phosphate and 1.584 g of disodium hydrogen phosphate) under sonication, adding 0.30 g of PVP and 9 mL of Tween 80 as surfactants. Then the obtained liposomal suspension was sonicated in an intelligent ultrasonic cell crushing machine (JYD-900; Zhixin Instrument, Co., Ltd., Shanghai, China) for 10 min. After free REOs and other impurities were removed by centrifugation at 2348.5 g × (Centrifuge 5424; Eppendorf, Germany), suspension of nanoliposomes containing REOs was obtained by filtering through a microporous membrane. Finally, the lyophilized nanoliposomes containing REOs, with relatively uniform distribution of particle size, were obtained by freeze-drying for 36 h with pre-freezing at 80 C for 8 h. Lyophilized nanoliposomes without REOs were prepared under − ◦ the same procedure.

2.3. Preparation of TSPU Solution The TSPU solution was synthesized using a two-step block copolymerization technique according to Zhou’s study [19]. Firstly, a mixture of 0.005 mol of PCL (Mw = 4000) and 0.010 mol of PEG (Mw = 2000) was mixed with 0.030 mol of MDI and reacted at 80 ◦C for 2 h to obtain TSPU pre-polymer. Additionally, DMF was added according to the viscosity of the reaction system, and the free isocyanate group (-NCO) was quantified by acetone-di-n-butylamine titration. Secondly, 0.060 mol of MDI and 0.0750 mol of BDO were added to the obtained TSPU pre-polymer for chain extension, and the reaction was carried out at 80 ◦C for 2 h with the ratio of -NCO and -OH fixed to 1:1, and final solid content controlled as 30% wt. Ultimately, a transparent and viscous TSPU solution was prepared.

2.4. Preparation of Complex Antioxidant Film Pure TSPU film was prepared by dry phase inversion technique. The prepared TSPU solution with solid content of 55.4 g was casted on a self-made rectangular rimmed polytetrafluoroethylene (PTEF) plate of 25 cm 15 cm, and dried at 60 C for 24 h in a DHG-9240A electro thermostatic blast oven × ◦ (Shanghai Jinghong Experimental Instrument Co., Ltd., Shanghai, China). The complex antioxidant film was prepared by coating TSPU solution incorporated with lyophilized nanoliposomes containing REOs onto BOPET through a solution casting technique, and identified as film A. The blank control film was prepared by coating TSPU solution without antioxidant onto BOPET, and identified as film B. The REO control film was prepared by coating TSPU solution directly incorporated with REOs instead of lyophilized nanoliposomes onto BOPET, and identified as film C. All the complex film (film A, B Materials 2019, 12, 4011 4 of 15 and C) were prepared with the same solid contents of TSPU (55.4 g), film area (25 cm 15 cm) and × heating condition (60 ◦C, 24 h) as pure TSPU film. To be comparable, the mass ratios of REOs to TSPU film in film A and film C were both controlled as 1%.

2.5. Quantification of REOs Qualitative and quantitative analysis of REOs was carried out by gas chromatography-mass spectrometry (GC-MS) with a HP-5 column (GCMS-QP2010Ultra, SHIMADZU, Japan). High pure helium was used as the carrier gas with flow rate of 1.0 mL/min. The detector was acquired by electron impact with scanning range of 33~600 m/z using an ionization energy of 70 eV. 1µL of sample was injected in the split ratio of 50:1. Temperatures of injector and detector were both 280 ◦C. The column was initially heated at 40 ◦C and remained 40 ◦C for 3 min, then heated at 5 ◦C/min to 100 ◦C and remained at 100 ◦C for 8 min, subsequently heated at 5 ◦C/min to 190 ◦C and remained at 190 ◦C for 5 min, lastly heated at 10 ◦C/min to 280 ◦C. Since 1, 8-eucalyptol was detected to be the main component of REO, the linear regression analysis of REO quantification was related to the peak area of 1, 8-eucalyptol in this study. For REOs dissolved in anhydrous ethanol and REOs volatized in the air, the determination coefficient (R2) of the linear regression analysis was 0.9992 and 0.9910, respectively. Specially, for REOs volatized in the air, the GC-MS detection required a pretreatment of solid phase micro-extraction (SPME) at 60 ◦C for 0.5 h.

2.6. Encapsulation Efficiency and Particle Characterization Encapsulation efficiency was determined by centrifugation techniques. In brief, the nanoliposomes in suspension and lyophilized nanoliposomes were dissolved in absolute ethanol, respectively. The encapsulated REOs were isolated by using centrifuge at 13527 g. Then the mass of REOs loaded in × nanoliposomes, identified as m1 (mg), was measured by GC-MS and calculated by linear regression 2 of which R = 0.9992. Moreover, the initially mass of the loaded REOs was identified as m0 (mg). Therefore, the encapsulation efficiency (EE) of the nanoliposomes was calculated by Equation (1) [20]. m EE = 1 100% (1) m0 ×

The mean diameter (MD) of particle, polydispersity index (PDI) and zeta potential of nanoliposomes in suspension and lyophilized nanoliposomes were measured by Malvern Zetasizer Nano (Malvern, Worcestershire, UK). Moreover, all of the above parameters were also measured at the 10th, 20th, 40th, and 60th day to evaluate the storage stability of nanoliposomes containing REOs at 4 ◦C.

2.7. Fourier Transform Infrared (FT-IR) Spectroscopy The FT-IR were obtained by using ALPHA-T Fourier transform infrared spectrometer (Bruker 1 Technology Co., Ltd, Beijing, China) with transmission mode, and scanning range of 600~4000 cm− . Prior to analysis, the samples were dried overnight.

2.8. Micro Structure Analysis The micro structure analysis was performed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). In the SEM test, for nanoliposomes, droplet samples of nanoliposome suspension were dropped onto a tin foil, after drying and spraying, the micro structure was observed and imaged using a SU1510 scanning electron microscope (HITACHI Ltd, Tokyo, Japan); while for films, the pretreatment was quenched in liquid nitrogen, attached to sample stub and then gold sprayed. AFM analysis was conducted through a MuLtimode8 atomic force microscope, and the pretreatment of nanoliposome suspension samples was performed by natural dry fixation. Materials 2019, 12, 4011 5 of 15

2.9. Differential Scanning Calorimetry (DSC) The DSC analysis was implemented using DSC Q2000 differential scanning calorimeter (Waters Co., Ltd, Milford, MA, USA). The test was performed under the protection of nitrogen, the temperature range was from 50 to 200 C, and the heating rate was 10 C/min. Each sample was tested twice, − ◦ ◦ the first test was to eliminate the thermal history of TSPU sample, and the second curve was used for analysis.

2.10. X-ray Diffraction (XRD) The crystallization performance analysis was obtained by X-ray diffractometer (D2 PHASER, Bruker AXS Ltd, Karlsruhe, Germany). The scanning range was 2θ = 5◦ to 40◦, the scanning speed was 0.1 s/step, and the scanning step size was 0.02◦.

2.11. Porosity Characterization The porosity analysis of the TSPU film used the quality loss method [21]. TSPU film sample was cut to suitable size. After being soaked in anhydrous ethanol, the wet film sample was weighed and the mass was identified as M2 (g). Then the wet film sample was dried until the quality remained constant, and the weight of dried film sample was identified as M1 (g). The ratio of the pore volume to the geometric volume of the TSPU film is identified as the porosity ε (%), which can be calculated by Equation (2). Conduct the tests at 15, 25, 35, 45, 55, and 65 ◦C, respectively.

M M ε = 2 − 1 100% (2) ρSd × where ρ is the density (g L 1) of anhydrous ethanol, S is the area (dm2), and d is the thickness (dm) of · − TSPU film sample, respectively.

2.12. Measure of Water Vapor Transmission Rate (WVTR) The water vapor permeability analysis of the TSPU film was conducted according to the ASTM E 96 standard [22]. Add equal amount of desiccant CaCl2 to 3 identical cups, respectively. Cover each cup with one piece of TSPU film and seal the cup to ensure that water vapor can only transport through the TSPU films. Then place the cups in a chamber with constant humidity of 50% RH, and constant temperature of 15, 25, 35, 45, 55, and 65 ◦C, respectively. Measure the mass change of CaCl2 in the cup after 24 h. Then, the water vapor transmission rate (WVPR) is calculated by Equation (3).

(W2 W ) 24 WVTR = − 1 × 100% (3) t S × × where W1 is the initial mass of CaCl2 added in the cup (g), W2 is the mass of CaCl2 in the cup after 24 h (g), t is the test time (h), S is the area of the cup (m2).

2.13. Release Characterization The one-way release experiments were carried out in a release installation as shown in Figure1 at 25, 40 and 60 ◦C, respectively. The one-way release was realized due to the high barrier of BOPET, thus REOs released only from the TSPU side of complex film to the air. Samples of film A and C (40 mm 40 mm) were placed into 15 mL brown , respectively. Sampling occurred every 3 days × with SPME method, and quantity of REO released was measured by GC-MS and calculated according to the linear regression with R2 = 0.9910. Materials 2019, 12, 4011 6 of 15 Materials 2019, 12, x FOR PEER REVIEW 6 of 15

FigureFigure 1. Schematic 1. Schematic diagram diagram of of release release installation. installation. A: polyethylenepolyethylene terephthalate terephthalate (PET); (PETB);: temperatureB: temperature sensitivesensitive polyurethane polyurethane (TSPU (TSPU);); C: CLyophilized: Lyophilized nanoliposomes nanoliposomes containing containing rosemary rosemary essential oilsoils (REOs).(REOs).

Finally, the diffusion coefficient (D) of REO release was calculated by Equation (4) based on Fick’s Finally, the diffusion coefficient (D) of REO release was calculated by Equation (4) based on Fick's secondsecond law law with with MATLAB MATLAB software software [23]. [23 ].  2  X 2 22 MMF,t ∞ 88  (212n + 1)π  Ft,= 1 exp   Dt (4) M 1 22 2 exp   2 Dt (4) F, − = (2n + 1) π2 − 4dp Md∞Fp, nn0 21n    4  where t is the diffusion time (s), Mt is the mass of the REO (mg) in the top space of at time t, M where t is the diffusion time (s), Mt is the mass of the REO (mg) in the top space of vials at time t, M∞ is∞ the is the mass of the REO (mg) in the top space of vials at time of equilibrium, d is the thickness (cm) of mass of the REO (mg) in the top space of vials at time of equilibrium, dp is the thicknessp (cm) of the composite 2 film,the and composite D is the diffusion film, and Dcoefficientis the diff (cmusion2/s)coe of REO.fficient (cm /s) of REO. If at the end of the experiment, the release of REO does not reach equilibrium (MF, t/MF, < 0.6), If at the end of the experiment, the release of REO does not reach equilibrium (MF, t/MF, ∞∞ < 0.6), the the diffusion coefficient D can be estimated by Equation (5) simplified from Equation (4). Where M diffusion coefficient D can be estimated by Equation (5) simplified from Equation (4). Where MF, p Fis, p the massis theof REO mass in of the REO original in the composite original composite film (mg). film In this (mg). study, In this Ds study, were Dcalculateds were calculated by Equation by Equation (5) for that (5) for that the release of REO does not reach equilibrium (MF, t/MF, < 0.6). the release of REO does not reach equilibrium (MF, t/MF, ∞ < 0.6). ∞ 1 1 M   2 F,t 4 Dt 2 M Ft, = 4 Dt (5) MF,p  dp π (5) Md F, p p 2.14. Antioxidant Activity

2.14. AntioxidantThe antioxidant Activity activity of REO-in-nanoliposomes and REO-in-nanoliposome-in-films were determined by DPPH radical scavenging activity assay, which is suitable for the analysis of antioxidant The antioxidant activity of REO-in-nanoliposomes and REO-in-nanoliposome-in-films were activity of botanical drug samples. For REO-in-nanoliposomes, 2 mL of suspension of nanoliposomes determined by DPPH radical scavenging activity assay, which is suitable for the analysis of antioxidant containing REOs was mixed with 2 mL of 100 µmol/L DPPH ethanol solution. The mixture was mixed activity of botanical drug samples. For REO-in-nanoliposomes, 2 mL of suspension of nanoliposomes vigorously and then kept in a dark at room temperature for 40 min. For REO-in-nanoliposome-in-films, containing REOs was mixed with 2 mL of 100 μmol/L DPPH ethanol solution. The mixture was mixed film A, B, and C of 40 mm 40 mm were cut into pieces and mixed with 2 mL of 100 µmol/L DPPH vigorously and then kept in a dark× at room temperature for 40 min. For REO-in-nanoliposome-in-films, film ethanol solution, respectively. The mixtures were mixed vigorously and then kept in dark at room A, B, and C of 40 mm × 40 mm were cut into pieces and mixed with 2 mL of 100 μmol/L DPPH ethanol temperature for 0.5 h, 24 h, 720 h and 1440 h, respectively. Control sample was obtained though solution, respectively. The mixtures were mixed vigorously and then kept in dark at room temperature for mixture of 2 mL of DPPH ethanol solution and 2 mL of pure ethanol solution. Then, the absorbance of 0.5 h, 24 h, 720 h and 1440 h, respectively. Control sample was obtained though mixture of 2 mL of DPPH mixture with REO and control sample were measured by UV-1800 spectrophotometer (SHIMADZU ethanol solution and 2 mL of pure ethanol solution. Then, the absorbance of mixture with REO and control Co., Ltd, Kyoto, Japan). The DPPH radical scavenging rate can be calculated by the Equation (6) [24]. sample were measured by UV-1800 spectrophotometer (SHIMADZU Co., Ltd, Kyoto, Japan). The DPPH radical scavenging rate can be calculated by the EquationAC (6)AS [24]. DPPHS(%) = − 100% (6) AC × AACS DPPH S %  100% (6) where DPPH is DPPH radical scavenging rate (%),AA is the absorbance of DPPH mixed with S C s nanoliposomes containing REOs, Ac is the absorbance of DPPH in control sample. where DPPHS is DPPH radical scavenging rate (%), As is the absorbance of DPPH mixed with nanoliposomes2.15. Statistical containing Analysis REOs, Ac is the absorbance of DPPH in control sample. The SPSS computer program (SPSS Inc., version 22) was used to carry out the one-way analysis of variance. Differences in pairs of mean values were evaluated by the Tukey test for a confidence interval of 95%. The data is presented as means standard deviation. ± Materials 2019, 12, x FOR PEER REVIEW 7 of 15 Materials 2019, 12, x FOR PEER REVIEW 7 of 15 2.15. Statistical Analysis 2.15. Statistical Analysis The SPSS computer program (SPSS Inc., version 22) was used to carry out the one-way analysis of variance.The DifferencesSPSS computer in pairs program of mean (SP valuesSS Inc., were version evaluated 22) was by usedthe Tukey to carry test out for thea confidence one-way intervalanalysis ofof Materials 2019, 12, 4011 7 of 15 95%.variance. The dataDifferences is presented in pairs as meansof mean ± standardvalues were deviation. evaluated by the Tukey test for a confidence interval of 95%. The data is presented as means ± standard deviation. 3. Results3. Results and andDiscussi Discussionon 3. Results and Discussion 3.1. 3.1.Characterization Characterization of Nanoliposomes of Nanoliposomes Containing Containing REOs REOs 3.1. Characterization of Nanoliposomes Containing REOs FT-IRFT-IR spectra spectra of of REO, REO, nanoliposomes nanoliposomes containing containing REOs REOs and and blank blank nanoliposomes nanoliposomes are are depicted in FigureinFT Figure 2a.-IR For spectra2 a.blank For ofnanoliposome, blank REO, nanoliposome, nanoliposomes the characteristic the containing characteristic absorption REOs absorption andpeaks blank are peaks mainly nanoliposomes are at mainly2924 and atare 2856 2924 depicted cm and−1 (C in– Figure 2a. For1 blank nanoliposome,−1 the characteristic1 absorption−1 peaks are mainly2− at1 2924 and 2856 cm2−1 (C– H, stretching2856 cm− vibration),(C–H, stretching 1737 cm vibration),(C=O, stretching), 1737 cm− 1245(C =cmO, stretching), (symmetrical 1245 PO cm stretching− (symmetrical vibration), PO 1103− H, −1stretching vibration), 1737 cm−11 (C=O,−1 stretching), 1245+ cm−1 (symmetrical1 PO2− stretching+ vibration), 1103 cm stretching (C–N, stretching), vibration), and 1103 948 cm cm− (C–N, (choline stretching), group andN CH 9483, cmstretching)− (choline [25]. group The NabsorptionCH3, stretching) bands of [25 REO]. cm−1 (C–N, stretching), and 948 cm−1 (choline group N+CH3, stretching)−1 [25]. The absorption bands1 of REO are Theat 2962 absorption–2881, 1745, bands 1464, of REO 1411, are 137 at6, 2962–2881, 1215, 1080 1745, and 1464,984 cm 1411,, due 1376, to 1215,the stretching 1080 and vibration 984 cm− ,of due C– toH, C are at 2962–2881, 1745, 1464, 1411, 1376, 1215, 1080 and−1 984 cm−1, due to the stretching vibration1 of C–H, C = O,the C = stretching C, O–H of vibration aromatic of rings C–H, (1411 C = O,and C 1376= C, cm O–H), ofO– aromaticH of CH rings2–OH, (1411 C–O andof –CH 13762–OH, cm− and), O–H bending of = O, C = C, O–H of aromatic rings (1411 and 1376 cm−1), O–H of CH2–OH, C–O of –CH2–OH, and bending vibrationCH2–OH, of C C–O–H, respectively of –CH2–OH, [26]. and With bending the addition vibration of of REO C–H, into respectively blank nanoliposome, [26]. With the most addition absorption of peaksvibrationREO of the into of REO C blank–H, loaded respectively nanoliposome, nanoliposome [26]. most With remain absorption the additionthe same, peaks of implying REO of the into REO that blank loadedno chemical nanoliposome, nanoliposome reaction most occurs remain absorption between the REOpeakssame, and of the implyingblank REO nanoliposome. loaded that no nanoliposome chemical Whilst reaction several remain occurs peaks the betweensame, shift implyingslightly REO and from that blank 2856no chemical nanoliposome. to 2858 reactioncm−1, 1737 Whilst occurs to several1735 between cm-1, REO and blank nanoliposome.−1 Whilst several 1peaks shift slightly-1 from 2856 to 2858 cm−1, 11737 to 1735 cm-1, andpeaks 1245 to shift 1247 slightly cm , respec fromtively, 2856 to indicating 2858 cm− that, 1737 there to are 1735 physical cm , andinteractions 1245 to 1247between cm− molecular, respectively, groups −1 ofand REOindicating 1245 and to 1247nanoliposome. that cm there, respec are physicaltively, indicating interactions that between there are molecular physical interactions groups of REO between and nanoliposome. molecular groups of REO and nanoliposome.

REO BOPET REO BOPET Nanoliposomes containing REOs TSPU Nanoliposomes containing REOs TSPU Blank nanoliposome A A Blank nanoliposome B B

C C

500 1000 1500 2000 2500 3000 3500 4000 500 1000 1500 2000 2500 3000 3500 4000 -1 500 1000 1500Wavenumbers/cm2000 2500 3000 3500 4000 500 1000 1500Wavenumbers/cm2000 2500 -13000 3500 4000 -1 Wavenumbers/cm Wavenumbers/cm-1 (a) (b) (a) (b) FigureFigure 2. FT 2.-IRFT-IR spectrogram spectrogram of REO, of REO, nanoliposomes nanoliposomes containing containing REOs REOs and blank and blank nanoliposomes nanoliposomes (a), and (a), FT- IRFigure spectrogramand 2. FT-IR FT-IR spectrogram ofspectrogram film A, B, of C, of film TSPUREO, A, B,nanoliposomesand C, BOPET TSPU and (b). containing BOPET (b). REOs and blank nanoliposomes (a), and FT- IR spectrogram of film A, B, C, TSPU and BOPET (b). SEMSEM and andAFM AFM images images of nanoliposomes of nanoliposomes containing containing REOs and REOs blank and nanoliposomes blank nanoliposomes are presented are in FigurepresentedSEM 3a. andIt can inAFM Figurebe observed images3a. Itof from can nanoliposomes be Figure observed 3a that containing from all Figurethe nanoliposomes REOs3a that and all blank the are nanoliposomes regular and smooth are presented regular spherical in vesiclesFigureand 3a. smoothwith It canuniform sphericalbe observed size and vesicles from homogen withFigureeous uniform 3a dispersion,that sizeall the and withoutnanoliposomes homogeneous obvious are aggregation dispersion, regular and withoutphenomenon. smooth obvious spherical vesiclesaggregation with uniform phenomenon. size and homogeneous dispersion, without obvious aggregation phenomenon.

(a) (b) (a) (b)

Figure 3. Cont. Materials 2019, 12, 4011 8 of 15 Materials 2019, 12, x FOR PEER REVIEW 8 of 15

(c) (d)

(e) (f)

FigureFigure 3. SEM 3. SEM images images of nanoliposomes of nanoliposomes containing containing REOs REOs (a) and (a) andblank blank nanoliposomes nanoliposomes (b), and (b), andAFM AFM images of nanoliposomesimages of nanoliposomes containing REOs containing (c,d) REOsand blank (c,d) nanoliposomes and blank nanoliposomes (e,f). (e,f).

TheThe EE,EE MD,, MD, PDI, PDI, Zeta Zeta potential potential and and antioxidant antioxidant activity activity results results of nanoliposomes of nanoliposomes containing containing REOs are REOs shown are in shown Table in 1. Table The1 . encapsulation The encapsulation efficiency e fficiency is an is important an important index index for for evaluating evaluating the the effect effect of encapsulating.of encapsulating. With aWith good a good repeatability, repeatability, the theEE EEof nanoliposomesof nanoliposomes freshly freshly prepared prepared is is 67.34%, 67.34%, and and the decreasethe decrease of EE ofis EE 0.82%is 0.82% after after 40 days 40 days of storage. of storage. The The results results present present an an effective effective encapsulation encapsulation and extraordinaryand extraordinary storage storagestability. stability. Normally, Normally, MD in the MD range in the of range 50 and of 50200 and nm 200 [27], nm PDI [27 between], PDI between 0 and 0.3 [28],0 and and absolute 0.3 [28], value and absolute of Zeta potential value of greater Zeta potential than 30 greatermV, means than a 30more mV, stable means and a moreuniform stable dispersion and of nanoliposome.uniform dispersion Moreover, of nanoliposome. the smaller Moreover, the PDI means the smaller more the concentrated PDI means particle more concentrated size and more particle stable system.size andIn th moreis research, stable system.MD, PDI, In and this Zeta research, potential MD, results PDI, and of nanoliposomes Zeta potential resultsfreshly ofprepared nanoliposomes and stored respectivelyfreshly prepared over 10, and 20, stored40 and respectively 60 days are over in the 10, range 20, 40 of and 60.75~64.57, 60 days are 0.230~0.236, in the range and of 60.75~64.57,−31.83~−30.02, 0.230~0.236, and 31.83~ 30.02, respectively. All the results are in the intervals that represent good respectively. All the− results − are in the intervals that represent good stability and uniform dispersion of nanoliposomestability and system. uniform The dispersion radical scavenging of nanoliposome rate of DPPH system. with The absorbance radical scavenging around 0.356 rate ofranges DPPH from 61.22%with to absorbance 55.77% with around a slight 0.356 decline ranges tendency from 61.22% as time to 55.77% consuming, with a slight indicating decline relatively tendency high as time radical scaveconsuming,nging activity. indicating Besides, relatively the relatively high radical minus scavenging change of activity. the above Besides, indexes the relativelyindicates excellent minus change storage stabilityof the over above 60 indexesdays. indicates excellent storage stability over 60 days.

Table 1. Table 1. EncapsulatingEncapsulating effect, effect, antioxidant antioxidant activity activity and and storage storage stability stability of nanoliposomes of nanoliposomes containing containing REOs REOs at 4 C. Means followed by the same letter in a column are not significantly different from each at 4°C. Means◦ followed by the same letter in a column are not significantly different from each other at P < other at P < 0.05. 0.05.

Time/dTime/d MD/nm MD/nm (s.(s. d.) d.) PDIPDI (s.(s. d.) d.) ZetaZeta Potential Potential/mV/mV (s.(s. d.) d.) EEEE/%/% (s.(s. d.)d.) DPPHsDPPHs/%/% (s.(s. d.) d.) a a a a a 0 60.75 a 1.079 0.230 a 0.0074 31.83 a 0.7767 67.34 a 1.649 61.22 a 1.427 0 60.75 1.079 0.230 0.0074 −−31.83 0.7767 67.34 1.649 61.22 1.427 10 61.15 a 2.734 0.231 a 0.0099 31.76 a 0.6737 67.21 a 4.695 60.57 a 1.484 10 61.15 a 2.734 0.231 a 0.0099 −−31.76 a 0.6737 67.21 a 4.695 60.57 a 1.484 20 61.23 a 2.314 0.231 a 0.0069 31.32 a 0.7263 66.70 ab 2.574 59.80 a 0.5814 a a − a ab a 2040 62.3961.23a 2.6582.314 0.2330.231a 0.00830.0069 −30.5631.32a 0.12270.7263 66.5266.70ab 0.41482.574 58.9359.80a 0.29980.5814 a a − a ab a 4060 64.5762.39a 1.222.658 0.2360.233a 0.00180.0083 −30.0230.56a 0.39980.1227 64.9266.52b 0.43440.4148 55.7758.93b 0.39760.2998 60 64.57 a 1.22 0.236 a 0.0018 −−30.02 a 0.3998 64.92 b 0.4344 55.77 b 0.3976 3.2. Double Switch Temperature Sensitive Characterization DSC curves of film A, B, C, TSPU are presented in Figure4a, where TSPU DSC curve shows 3.2.two Double phase Switch transition Temperature temperature Sensitive (crystalline Characterization and melting peaks) of 35.26 ◦C and 56.98 ◦C, caused by temperature sensitive property of PEG2000 and PCL4000 soft segment, respectively, indicating a smart Materials 2019, 12, x FOR PEER REVIEW 9 of 15

Materials 2019, 12, 4011 9 of 15 DSC curves of film A, B, C, TSPU are presented in Figure 4a, where TSPU DSC curve shows two phase transition temperature (crystalline and melting peaks) of 35.26 °C and 56.98 °C, caused by membranetemperature with sensitive double temperature property of PEG2000 switch is and acquired. PCL4000 There soft are segment, also two respectively, similar phase indicating transition a smart membrane with double temperature switch is acquired. There are also two similar phase temperature (crystalline melting peaks) of film A (39.56 ◦C, 56.00 ◦C), film B (36.04 ◦C, 57.32 ◦C), and transition temperature (crystalline melting peaks) of film A (39.56 °C, 56.00 °C), film B (36.04 °C, 57.32 film C (35.26 ◦C, 57.49 ◦C), respectively. The results demonstrate that the coating of TSPU onto BOPET °C), and film C (35.26 °C, 57.49 °C), respectively. The results demonstrate that the coating of TSPU (film B), the addition of REO (film C) and the incorporation of nanoliposomes containing REOs (film onto BOPET (film B), the addition of REO (film C) and the incorporation of nanoliposomes containing A) remain the double-switch temperature-sensitive characteristic of TSPU film obtained in this study. REOs (film A) remain the double-switch temperature-sensitive characteristic of TSPU film obtained In addition, compared with the acquired TSPU, the lower phase transition temperature of film A in this study. In addition, compared with the acquired TSPU, the lower phase transition temperature increasesof film slightlyA increases from slightly 35.26 tofrom 39.56 35.26◦C. to This 39.56 could °C. beThis attributed could be toattributed the enhanced to the rigidityenhanced and rigidity limited motionand limited of PEG2000 motion soft of segmentPEG2000 resulting soft segment from resulting the interaction from the between interaction nanoliposomes between nanoliposomes and PEG2000 softand segment. PEG2000 soft segment.

(a) (b)

） TSPU

W/g

（ A C B B A

C Intensity/a.u. Heat flow/ TSPU BOPET -50 0 50 100 150 200 0 5 10 15 20 25 30 35 40 45 Temperature/℃ 2θ/° Figure 4. DSC curves of film A, B, C, TSPU (a), and XRD patterns of film A, B, C, TSPU, BOPET (b).

XRDFigure patterns 4. DSC of curves film A, of B, film C, TSPUA, B, C, and TSPU BOPET (a), and are XRD depicted patterns in of Figure film A,4 b.B, C, XRD TSPU, pattern BOPET of ( TSPUb). has peaks of 2θ mainly at 21.6◦, 22.2 and 23.9◦ which correspond to the crystalline peaks of the soft segment and theXRD hard patterns segment of [film29], A, indicating B, C, TSPU that and the BOPET hard are segment depicted MDI in Fig doesure not 4b. destroyXRD pattern the crystallineof TSPU morphologyhas peaks ofof the2θ mainly soft segment at 21.6°, PEG 22.2 and and PCL. 23.9° There which are correspond also two similar to the dominantcrystalline characteristic peaks of the soft peaks segment and the hard segment [29], indicating that the hard segment MDI does not destroy the of 2θ of film A (21.7◦, 24.0◦), film B (21.9◦, 24.2◦), and film C (21.9◦, 24.1◦), respectively. The results implycrystalline that coating morphology of TSPU of onto the BOPET soft segment (film B), PEG the and addition PCL. There of REO are (film also C) two and similar the incorporation dominant of nanoliposomescharacteristic peaks containing of 2θ of REOs film (film A (21.7°, A) does 24.0°), not film disrupt B (21.9°, the intrinsic 24.2°), and crystalline film C morphology (21.9°, 24.1°), of TSPUrespectively. film obtained The results in this study.imply that This coating is in accordance of TSPU onto with BOPET DSC results (film B), that the the addition TSPU basedof REO complex (film C) and the incorporation of nanoliposomes containing REOs (film A) does not disrupt the intrinsic films still have temperature response characteristics. Furthermore, compared with pure TSPU film, the crystalline morphology of TSPU film obtained in this study. This is in accordance with DSC results peak intensity of film A, B and C all decrease apparently, demonstrating a decrease of crystallinity of that the TSPU based complex films still have temperature response characteristics. Furthermore, the soft segment or hard segment. Besides, all the BOPET based films have similar peak of 2θ at 26.0 compared with pure TSPU film, the peak intensity of film A, B and C all decrease apparently, ◦ (film A), 26.0 (film B) and 26.1 (film C) compared with BOPET (26.9 ), implying that film A, B and C demonstrating◦ a decrease of c◦rystallinity of the soft segment or hard segment.◦ Besides, all the BOPET remainbased the films crystalline have similar morphology peak of 2 ofθ at pure 26.0° BOPET (film A), film. 26.0° Moreover, (film B) withand 26.1° increase (film of C) peak compared intensity, with the crystallinityBOPET (26.9°), of film implying A, B and that C film increase A, B and compared C remain with the pure crystalline BOPET morphology film. of pure BOPET film. Moreover,Porosity withε and increaseWVTR of peak TSPU intensity, and PU filmsthe crystallinity at different of temperatures film A, B and rangingC increase from compared 15 ◦C to with 65 ◦ C arepure described BOPET in film. Figure 5a,b, respectively. The diagram shows that both porosity and WVTR of the control filmPorosity PUwithout ε and WVTR temperature of TSPU sensibility and PU films rise at slightly different as regulartemperatures with the ranging increase from of 15 temperature. °C to 65 However,°C are described the porosity in Figure and 5a,b,WVTR respectively.of TSPU film The undergodiagram shows two irregular that both significant porosity and increase WVTR aroundof the phasecontrol transition film PU temperature without oftemperature 35.26 ◦C and sensibility 56.98 ◦C. rise Specifically, slightly theas regular porosity with of TSPU the increase increase fromof 19.81%temperature. at 25 ◦C to However, 33.67% at the 35 ◦ porosityC and from and48.49% WVTR atof 55 TSPU◦C to film 68.24% undergo at 65 ◦ C, two showing irregular two significant significant improvementsincrease around as high phase as 170%transition and temperature 141%, respectively. of 35.26 Consistent °C and 56.98 with °C. the Specifically, porosity increase the porosity tendency, of 2 2 theTSPUWVTR increaseof TSPU from increase 19.81% at from 25 °C 70.49 to 33.67% g/m 24 at 35 h at°C 25 and◦C fro tom 145.71 48.49% g /atm 5524 °C h to at 68.24% 35 ◦C at and 65 °C, from 2 2 · · 306.32showing g/m two24 h significant at 55 ◦C to improvements 578.54 g/m 24 as hhigh at 65 as◦ C,170% showing and 141%, two respectively. significant improvements Consistent with as the high · · 2 2 as 207%porosity and increase 189%, tendency, respectively. the WVTR The results of TSPU indicate increase that from with 70.49 the g/m phase·24 h transition at 25 °C to of 145.71 PEG2000 g/m ·24 and PCL4000 soft segment in TSPU controlled by temperature, the porosity representing the size of the free volume hole in TSPU and micro-Brownian motion in the polymer [30] alters rigorously, thus the WVTR of TSPU changes in the same tendency. In this , WVTR of TSPU determined by porosity illustrates Materials 2019, 12, x FOR PEER REVIEW 10 of 15

h at 35 °C and from 306.32 g/m2·24 h at 55 °C to 578.54 g/m2·24 h at 65 °C, showing two significant improvements as high as 207% and 189%, respectively. The results indicate that with the phase

Materialstransition2019, 12 of, 4011 PEG2000 and PCL4000 soft segment in TSPU controlled by temperature, the porosity10 of 15 representing the size of the free volume hole in TSPU and micro-Brownian motion in the polymer [30] alters rigorously, thus the WVTR of TSPU changes in the same tendency. In this case, WVTR of thatTSPU the TSPU determined film fabricated by porosity in thisillustrates study hasthat anthe intelligent TSPU film temperature fabricated in responsethis study characteristic has an intelligent with a doubletemperature switch. response characteristic with a double switch.

(a) 70 (b) 600 60 500

50 -1 400

·d

40 -2

m 300 30 200 Porosity/% 20 PU 100 WVTR/g· PU TSPU 10 TSPU 0 10 20 30 40 50 60 70 10 20 30 40 50 60 70 Temperature/℃ Temperature/℃ Figure 5. Porosity of TSPU and PU films (a), and WVTRs of TSPU and PU films (b).

3.3. CharacterizationFigure of 5. Controlled Porosity of ReleaseTSPU and Complex PU films Film (a), and WVTRs of TSPU and PU films (b). Figure2b is the FT-IR spectra of film A, B, C, TSPU, and BOPET. For TSPU, absorption peak at 3.3. Characterization1 of Controlled Release Complex Film 3311 cm− assigns to the stretching vibration of hydroxyl group O–H and asymmetric and symmetric 1 stretchingFigure of N–H, 2b is the peak FT at-IR 2946 spectra and of 2867 film cmA, B,− C,assigns TSPU, toand the BOPET. bending For and TSPU, stretching absorption vibration peak at of −1 1 C–H,3311 peaks cm assigns at 1537, to 1305, the stretching 1237, 1105, vibration 1019 andof hydroxyl 952 cm group− correspond O–H and toasymmetric the bending and vibrationsymmetric of stretching of N–H, peak at 2946 and 2867 cm−1 assigns to the bending1 and stretching vibration of C– N–H, stretching vibration of O–H of –CH2–OH (1305 and 1237 cm− ), stretching of C–N, C–O, and bendingH, peaks vibration at 1537, of 1305,=C–H, 1237, respectively. 1105, 1019 and There 952 is cm no−1 characteristic correspond to absorption the bending peak vibration of –NCO of N in–H, the stretching vibration of1 O–H of –CH2–OH (1305 and 1237 cm−1), stretching1 of C–N, C–O, and bending range of 2260–2280 cm− , while an absorption peak appears at 1725 cm− , indicating that carbamate groupvibration (-NHCOO-) of =C–H, has res beenpectively. produced There through is no characteristic the nucleophilic absorption reaction peak between of –NCO –NCO in the and range –OH of [8 ]. 2260-2280 cm−1, while an absorption peak appears at 1725 cm−1, indicating that carbamate group (- The incorporation of REO loaded lyophilized nanoliposomes into TSPU does not alter most of the NHCOO-) has been produced through the nucleophilic reaction between –NCO and –OH [8]. The characteristic peak of TSPU except minus shifts of 3311 to 3313 cm 1, 1305 to 1307 cm 1, 1237 to incorporation of REO loaded lyophilized nanoliposomes into TSPU− does not alter most− of the 1227 cm 1, and 952 to 950 cm 1, respectively. This reveals that no chemical bond is newly built characteristic− peak of TSPU except− minus shifts of 3311 to 3313 cm−1, 1305 to 1307 cm−1, 1237 to 1227 up between REO loaded lyophilized nanoliposomes and TSPU, while between which exists a weak cm−1, and 952 to 950 cm−1, respectively. This reveals that no chemical bond is newly built up between physicalREO loaded interaction. lyophilized In the contrast, nanoliposomes the direct and incorporation TSPU, while betweenof REO into which TSPU exists dramatically a weak physical changes 1 1 someinteraction. of the characteristic In the contrast, peaks the direct of TSPU. incorporation The peak of at REO 3311 into cm TSPU− not dramatically only shifts tochanges 3323 cmsome− , of but 1 alsothe substantially characteristi decreasesc peaks of the TSPU. intensity The of peak peak. at 3311The peaks cm−1 not at 1305, only 1019 shifts and to 3323952 cm cm−−1,also but have also a 1 relativelysubstantially large scaled decreases shift the to 1294, intensity 1046 andof peak. 960 cm The− . peaks The results at 1305, indicate 1019 andthat the952 physical cm−1 also interaction have a betweenrelatively REO large and TSPUscaled is shift stronger to 1294, than 1046 which and between 960 cm−1 lyophilized. The results nanoliposomes indicate that encapsulating the physical REOinteraction and TSPU. between REO and TSPU is stronger than which between lyophilized nanoliposomes encapsulatingSEM pictures REO performing and TSPU. surface micromorphology of TSPU side and cross-section of film A, B and C areSEM exhibited pictures inperforming Figure6. s Iturface can bemicromorphology observed from of the TSPU TSPU side side and surface cross-section images of that film film A, B B as blankand C controlare exhibited film is in relatively Figure 6. denseIt can be and observed smooth, from while the film TSPU A incorporatedside surface images with nanoliposomesthat film B as containingblank control REOs film is slightly is relatively uneven dense with and aggregated smooth, nanoliposomeswhile film A incorporated and small amountwith nanoliposomes of micropores, andcontaining film C as REOs REO controlis slightly film uneven has more with microporesaggregated nanoliposomes compared with and film small A. The amount micropores of micro mightpores, be formedand film by the C as volatilization REO control offilm REO has during more micropores the film drying compared process. with Moreover, film A. The the micropores less micropores might of filmbe A formed compared by the with volatilization film C is probably of REO during due to the the film decline drying of process. REO volatilization, Moreover, the resulting less micropores from the reinforcedof film A interaction compared with between film theC is nanoliposomesprobably due to andthe decline the soft of segment REO volatilization, in TSPU film resulting in agreement from withthe DSC reinforced results in interaction this study between (Section the 3.2 nanoliposomes). Likewise, the and amount the soft of micropores segment in presented TSPU film in in the agreement with DSC results in this study (Section 3.2). Likewise, the amount of micropores presented cross-section image of film A, B and C obey the same rule as in the TSPU side surface image, while the distribution of micropores is more in the center and less on the surface. In addition, the cross-section morphology of all the three films present a good consistency without delamination at the interface of TSPU and BOPET, indicating that the TSPU layer is well bonded to the BOPET layer. Materials 2019, 12, x FOR PEER REVIEW 11 of 15

in theMaterials cross 2019-sectio, 12, xn FOR image PEER ofREVIEW film A, B and C obey the same rule as in the TSPU side surface11 image, of 15 while the distribution of micropores is more in the center and less on the surface. In addition, the crossin -thesection cross morphology-section image of of all film the A, three B and films C obey present the samea good rule consistency as in the TSPU without side delaminationsurface image, at while the distribution of micropores is more in the center and less on the surface. In addition, the the interface of TSPU and BOPET, indicating that the TSPU layer is well bonded to the BOPET layer. Materialscross2019-section, 12, 4011 morphology of all the three films present a good consistency without delamination11 ofat 15 the interface of TSPU and BOPET, indicating that the TSPU layer is well bonded to the BOPET layer.

(a) (b) (c) (a) (b ) (c)

(d) (e) (f) (d) (e) (f)

FigureFigure 6.6. SEM6. SEM surfacesurface surface picturespictures pictures of of TSPUof TSPU TSPU side side side of of filmof film film A, A,A, B andBB and C (Ca– (ca),––c andc),), andand cross-section crosscross-section-section of of film of film film A, A, B A, andB B C(anddand –Cf ),( Cd respectively.– (fd),– frespectively.), respectively.

BothBoth the the test test data data and and fittingfitting fitting curves curves of of REO REO releasingreleasing proportion outout of of film film film C C and and A A at at 25 25 °C, °C,◦ C, 40 ◦°C40C °C and and 6060 60◦°CC °C areare are depicteddepicted depicted inin in FigureFigure Figure7 a,b,7a,b 7a,b respectively., ,respectively. respectively. The The goodness goodness of ofof fit fit fit is is evaluatedis evaluated evaluated by by by means means means of theof thof roote th roote meanroot mean mean square square square error error error (RMSE) (RMSE) (RMSE) listed listed listed in in in Table Table Table2. 2.2. The TheThe results results show showshow thatthat that thethe the model model satisfactorily satisfactorily satisfactorily fitsfitsfits the the test test data, data, suggestingsuggesting suggesting the the adopted adopted model model isis suitablesuitable for characterization of of REO REO release release from from thethe complex complex film.film. film. The The The calculated calculated calculated DD Dss sare are are presented presented in in Tab TableTable 22,, andandand thethethe increaseincreaseincrease tendency tendencytendency of ofof D D with with temperaturetemperature ascendingascending ascending isis is describeddescribed described inin in FigureFigure Figure7 7c. c.7c.

Figure 7. Cont. Materials 2019, 12, 4011 12 of 15 Materials 2019, 12, x FOR PEER REVIEW 12 of 15

Figure 7. Test data and fitting curves of REO release from film C (a) and film A (b) at 25, 40 and 60 ◦C, Figurerespectively, 7. Test and dataD andof film fitting C and curves A at of 25, REO 40 andrelease 60 ◦fromC, respectively film C (a) and (c). film A (b) at 25, 40 and 60 °C, respectively,Table 2. Diff usionand D coe of filmfficient C andD of A REO at 25, release 40 and from 60 °C, film respectively A and C at (c di). fferent temperatures. Means followed by the same letter in a column are not significantly different from each other at P < 0.05. Table 2. Diffusion coefficient D of REO release from film A and C at different temperatures. Means 14 2 1 followed by the same letter in a column are not significantlyD/ 10− different(cm s− ) from each other at P < 0.05. Film Samples × · 25 ◦C RMSED 40/×10◦C−14 (cm2·s RMSE−1) 60 ◦C RMSE Film Samples a a a A 1.212 25 °C0.001220 RMSE 1.64840 °C RMSE0.001509 60 °C 2.069RMSE 0.001967 a a a CA 1.627 1.2120.002744 a 0.001220 2.3061.648 a 0.0015090.003596 2.069 a 2.7020.001967 0.004054 C 1.627 a 0.002744 2.306 a 0.003596 2.702 a 0.004054 It can be observed that the release of REO from both films (film C and A) at all temperatures It can be observed that the release of REO from both films (film C and A) at all temperatures (25 (25 ◦C, 40 ◦C and 60 ◦C) has not reached equilibrium in 60 days, and the mass rate of released REO °C,was 40 less °C thanand 60 10%, °C) indicating has not reach a veryed equilibrium slow release in of 60 REO. days, This and is the in accordancemass rate of withreleased the lowREO value was less than 10%, indicating a14 very 2slow1 release of REO. This is in accordance with the low value of D at of D at magnitude of 10− (cm s− ). Moreover, Ds of film A are smaller than that of film C at each −14 2 −1 · magnitudetemperature of tested,10 (cm implying·s ). Moreover, a significant Ds of barrier film A and are smaller protection than eff thatect ofof nanoliposomefilm C at each temperature layer in this tested,study. Although implying D a s significant of film C are barrier larger and than protectionDs of film effect A at each of nanoliposome temperature tested, layer in the this relatively study. Although Ds of film C are larger than Ds of film A at each temperature14 2 1 tested, the relatively low low release rate of REO represented by D at magnitude of 10− (cm s− ) is probably caused by the −14 2 −1 · releasestrong interactionrate of REO betweenrepresented directly by D addedat magnitude REO and of 10 TSPU (cm as·s discussed) is probably before caused according by the to strong FT-IR interactionresults. Furthermore, between directly with the added temperature REO and ascending, TSPU asD discussedof both film before C and according A undergoes to FT an-IR abnormal results. Furthermore,dramatically increase with the (Figure temperature7c) against ascending, Arrhenius D of theory, both film in which C andD Ais undergoes exponentially an abnormalrelated to dramaticallytemperature. increase This demonstrates (Figure 7c) thatagainst both Arrhenius complex filmtheory, C and in which A have D remained is exponentially the double related switch to temperature.temperature sensitiveThis demonstrates property ofthat TSPU, both and complex boost thefilm release C and aroundA have phaseremained transition the double temperature switch temperature sensitive property of TSPU, and boost the release around phase transition temperature 39.56 ◦C and 56.00 ◦C. The intelligent temperature-sensitive controlled release function is realized via 39.56enlarging °C and the 56.00 free volume°C. The holeintelligent size and temperature accelerating-sensitive micro-Brownian controlled motion release of function soft segment is realized in TSPU. via enlargingThe results the free of volume the DPPH hole radical size and scavenging accelerating test micro for film-Brownian A, B and motion C are shown of soft in segment Table3. in It canTSPU. be concludedThe results from Tableof the3 DPPHthat the radical DPPH scavenging radical scavenging test for film activity A, B of and film C A are gradually shown in increases Table 3. during It can bethe concluded period of 1440from h Table (60 d). 3 Thethat extremelythe DPPH low radicalDPPHs scavengingof film B mightactivity be of caused film A by gradually experimental increases error. duringFor film the C, the period DPPH of free 1440 radical h (60 scavenging d). The extremely activity is low higher DPPHs within of 720 film h B (30 might d) whereas be caused lower by at experimental1440 h (60 d) comparederror. For withfilm filmC, the A. DPPH The results free radical demonstrate scavenging that film activit A hasy is a lastinghigher antioxidantwithin 720 h eff (30ect d)due whereas to the controlled lower at 1440 release h (60 property d) compared of nanoliposomes. with film A. Besides,The results the obtaineddemonstrate multicomponent that film A has film a lastingcan be recycled antioxidant bysolving effect due isolation to the method controlled according release to research property of of Patrizia nanoliposomes. et al. [31]. Besides, the obtained multicomponent film can be recycled by solving isolation method according to research of Patrizia et al. [31] Materials 2019, 12, 4011 13 of 15

Table 3. DPPH radical scavenging activity of film A, B and C. Means followed by the same letter in a column are not significantly different from each other at P < 0.05.

Absorbance DPPHs/% Film Samples 0.5 h (s. d.) 24 h (s. d.) 720 h (s. d.) 1440 h (s. d.) 0.5 h (s. d.) 24 h (s. d.) 720 h (s. d.) 1440 h (s. d.) Control 0.918 a 0.0223 0.918 a 0.0026 0.917 a 0.0066 0.917 a 0.0194 0.000 a 0.0000 0.000 a 0.0000 0.000 a 0.0000 0.000 a 0.0000 A 0.585 b 0.0142 0.525 b 0.0017 0.481 b 0.0067 0.435 b 0.0085 36.27 b 0.8533 42.81 b 0.2892 47.55 b 1.663 52.56 b 0.1818 B 0.904 a 0.0019 0.906 a 0.009 0.902 a 0.0246 0.901 a 0.0637 1.530 a 0.0055 1.300 c 0.0015 1.640 a 0.0055 1.740 c 0.0122 C 0.512 c 0.0178 0.463 c 0.0328 0.472 b 0.0028 0.526 b 0.0251 44.23 c 1.019 49.57 d 0.6615 48.53 b 1.01 42.64 d 0.2782 Materials 2019, 12, 4011 14 of 15

4. Conclusions

In this study, a TSPU film with double switch temperature at 35.26 ◦C and 56.98 ◦C was prepared by copolymerization of PEG2000 and PCL4000 soft segment. With BOPET as a barrier layer, an intelligent double-switch temperature-sensitive controlled release antioxidant film was fabricated via coating the prepared TSPU embedded with lyophilized nanoliposomes encapsulating REOs on BOPET. The results indicate that the REO is well encapsulated in nanoliposomes with EE of 67.34%, high stability and lasting antioxidant effect during 60 days. The incorporation of nanoliposomes containing REOs into TSPU does not destroy the double-switch temperature-sensitive characteristic of the prepared TSPU. In agreement with porosity and WVTR results, the diffusion coefficient D of the antioxidant complex film sharply increases respectively at two switching temperatures, indicating that the intelligent double switch temperature-sensitive controlled release property is functioning. Furthermore, compared with films directly added with REO, the lower Ds of films added with lyophilized nanoliposomes encapsulating REOs provides a longer-lasting antioxidant activity. In general, the acquired controlled release antioxidant film sensitive to temperature at 39.56 ◦C and 56.00 ◦C can be potentially applied for the protection of solid food during the distribution and storage process under severe high temperatures.

Author Contributions: Investigation, X.C., Q.L.; Methodology, L.-X.L., L.Z.; Project administration, L.Z., L.-X.L.; Resources, L.P., L.-J.L., W.-R.Y.; Writing—original draft, X.C., Q.L., L.-N.S. Funding: This research was funded by National Key R&D Program, grant number 2016YFD0400701 and 2018YFC1603202/2018YFC1603200. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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