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

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 polyethylene 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.


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

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 (M w = 4000) and 0.010 mol of PEG (M w = 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.

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 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%.

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 (R 2 ) 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.

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 m 1 (mg), was measured by GC-MS and calculated by linear regression of which R 2 = 0.9992. Moreover, the initially mass of the loaded REOs was identified as m 0 (mg). Therefore, the encapsulation efficiency (EE) of the nanoliposomes was calculated by Equation (1) [20].
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.

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

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 gold 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.

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.

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 • .

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 M 2 (g). Then the wet film sample was dried until the quality remained constant, and the weight of dried film sample was identified as M 1 (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.
where ρ is the density (g·L −1 ) of anhydrous ethanol, S is the area (dm 2 ), and d is the thickness (dm) of TSPU film sample, respectively.

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 CaCl 2 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 CaCl 2 in the cup after 24 h. Then, the water vapor transmission rate (WVPR) is calculated by Equation (3).
where W 1 is the initial mass of CaCl 2 added in the cup (g), W 2 is the mass of CaCl 2 in the cup after 24 h (g), t is the test time (h), S is the area of the cup (m 2 ).

Release Characterization
The one-way release experiments were carried out in a release installation as shown in Figure 1 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 vial, 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 R 2 = 0.9910.  Finally, the diffusion coefficient (D) of REO release was calculated by Equation (4) 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 mass of the REO (mg) in the top space of vials at time of equilibrium, dp is the thickness (cm) of the composite film, and D is the diffusion coefficient (cm 2 /s) of REO.
If at the end of the experiment, the release of REO does not reach equilibrium (MF, t/MF, ∞ < 0.6), the diffusion coefficient D can be estimated by Equation (5) simplified from Equation (4). Where MF, p is the mass of REO in the original composite film (mg). In this study, Ds were calculated by Equation (5) for that the release of REO does not reach equilibrium (MF, t/MF, ∞ < 0.6).

Antioxidant Activity
The antioxidant 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 activity of botanical drug samples. For REO-in-nanoliposomes, 2 mL of suspension of nanoliposomes containing REOs was mixed with 2 mL of 100 μmol/L DPPH ethanol solution. The mixture was mixed vigorously and then kept in a dark at room temperature for 40 min. For REO-in-nanoliposome-in-films, film A, B, and C of 40 mm × 40 mm were cut into pieces and mixed with 2 mL of 100 μmol/L DPPH ethanol solution, respectively. The mixtures were mixed vigorously and then kept in dark at room temperature for 0.5 h, 24 h, 720 h and 1440 h, respectively. Control sample was obtained though mixture of 2 mL of DPPH ethanol solution and 2 mL of pure ethanol solution. Then, the absorbance of mixture with REO and control sample were measured by UV-1800 spectrophotometer (SHIMADZU Co., Ltd, Kyoto, Japan). The DPPH radical scavenging rate can be calculated by the Equation (6) [24].
where DPPHS is DPPH radical scavenging rate (%), As is the absorbance of DPPH mixed with nanoliposomes containing REOs, Ac is the absorbance of DPPH in control sample. Finally, the diffusion coefficient (D) of REO release was calculated by Equation (4) based on Fick's second law with MATLAB software [23].
where t is the diffusion time (s), M t is the mass of the REO (mg) in the top space of vials at time t, M ∞ is the mass of the REO (mg) in the top space of vials at time of equilibrium, d p is the thickness (cm) of the composite film, and D is the diffusion coefficient (cm 2 /s) of REO.
If at the end of the experiment, the release of REO does not reach equilibrium (M F, t /M F, ∞ < 0.6), the diffusion coefficient D can be estimated by Equation (5) simplified from Equation (4). Where M F, p is the mass of REO in the original composite film (mg). In this study, Ds were calculated by Equation (5) for that the release of REO does not reach equilibrium (M F, t /M F, ∞ < 0.6).

Antioxidant Activity
The antioxidant 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 activity of botanical drug samples. For REO-in-nanoliposomes, 2 mL of suspension of nanoliposomes containing REOs was mixed with 2 mL of 100 µmol/L DPPH ethanol solution. The mixture was mixed vigorously and then kept in a dark at room temperature for 40 min. For REO-in-nanoliposome-in-films, film A, B, and C of 40 mm × 40 mm were cut into pieces and mixed with 2 mL of 100 µmol/L DPPH ethanol solution, respectively. The mixtures were mixed vigorously and then kept in dark at room temperature for 0.5 h, 24 h, 720 h and 1440 h, respectively. Control sample was obtained though mixture of 2 mL of DPPH ethanol solution and 2 mL of pure ethanol solution. Then, the absorbance of mixture with REO and control sample were measured by UV-1800 spectrophotometer (SHIMADZU Co., Ltd, Kyoto, Japan). The DPPH radical scavenging rate can be calculated by the Equation (6) [24].
where DPPH S is DPPH radical scavenging rate (%), A s is the absorbance of DPPH mixed with nanoliposomes containing REOs, A c is the absorbance of DPPH in control sample.

Statistical Analysis
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.

Characterization of Nanoliposomes Containing REOs
FT-IR spectra of REO, nanoliposomes containing REOs and blank nanoliposomes are depicted in Figure [26]. With the addition of REO into blank nanoliposome, most absorption peaks of the REO loaded nanoliposome remain the same, implying that no chemical reaction occurs between REO and blank nanoliposome. Whilst several peaks shift slightly from 2856 to 2858 cm −1 , 1737 to 1735 cm -1 , and 1245 to 1247 cm −1 , respectively, indicating that there are physical interactions between molecular groups of REO and nanoliposome.

Statistical Analysis
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.

Characterization of Nanoliposomes Containing REOs
FT-IR spectra of REO, nanoliposomes containing REOs and blank nanoliposomes are depicted in Figure [26]. With the addition of REO into blank nanoliposome, most absorption peaks of the REO loaded nanoliposome remain the same, implying that no chemical reaction occurs between REO and blank nanoliposome. Whilst several peaks shift slightly from 2856 to 2858 cm −1 , 1737 to 1735 cm -1 , and 1245 to 1247 cm −1 , respectively, indicating that there are physical interactions between molecular groups of REO and nanoliposome.

Statistical Analysis
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.

Characterization of Nanoliposomes Containing REOs
FT-IR spectra of REO, nanoliposomes containing REOs and blank nanoliposomes are depicted in  [26]. With the addition of REO into blank nanoliposome, most absorption peaks of the REO loaded nanoliposome remain the same, implying that no chemical reaction occurs between REO and blank nanoliposome. Whilst several peaks shift slightly from 2856 to 2858 cm −1 , 1737 to 1735 cm -1 , and 1245 to 1247 cm −1 , respectively, indicating that there are physical interactions between molecular groups of REO and nanoliposome.  The EE, MD, PDI, Zeta potential and antioxidant activity results of nanoliposomes containing REOs are shown in Table 1. The encapsulation efficiency is an important index for evaluating the effect of encapsulating. With a good repeatability, the EE of nanoliposomes freshly prepared is 67.34%, and the decrease of EE is 0.82% after 40 days of storage. The results present an effective encapsulation and extraordinary storage stability. Normally, MD in the range of 50 and 200 nm [27], PDI between 0 and 0.3 [28], and absolute value of Zeta potential greater than 30 mV, means a more stable and uniform dispersion of nanoliposome. Moreover, the smaller the PDI means more concentrated particle size and more stable system. In this research, MD, PDI, and Zeta potential results of nanoliposomes freshly prepared and stored respectively over 10, 20, 40 and 60 days are in the range of 60.75~64.57, 0.230~0.236, and −31.83~−30.02, respectively. All the results are in the intervals that represent good stability and uniform dispersion of nanoliposome system. The radical scavenging rate of DPPH with absorbance around 0.356 ranges from 61.22% to 55.77% with a slight decline tendency as time consuming, indicating relatively high radical scavenging activity. Besides, the relatively minus change of the above indexes indicates excellent storage stability over 60 days.  The EE, MD, PDI, Zeta potential and antioxidant activity results of nanoliposomes containing REOs are shown in Table 1. The encapsulation efficiency is an important index for evaluating the effect of encapsulating. With a good repeatability, the EE of nanoliposomes freshly prepared is 67.34%, and the decrease of EE is 0.82% after 40 days of storage. The results present an effective encapsulation and extraordinary storage stability. Normally, MD in the range of 50 and 200 nm [27], PDI between 0 and 0.3 [28], and absolute value of Zeta potential greater than 30 mV, means a more stable and uniform dispersion of nanoliposome. Moreover, the smaller the PDI means more concentrated particle size and more stable system. In this research, MD, PDI, and Zeta potential results of nanoliposomes freshly prepared and stored respectively over 10, 20, 40 and 60 days are in the range of 60.75~64.57, 0.230~0.236, and −31.83~−30.02, respectively. All the results are in the intervals that represent good stability and uniform dispersion of nanoliposome system. The radical scavenging rate of DPPH with absorbance around 0.356 ranges from 61.22% to 55.77% with a slight decline tendency as time consuming, indicating relatively high radical scavenging activity. Besides, the relatively minus change of the above indexes indicates excellent storage stability over 60 days.  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 temperature sensitive property of PEG2000 and PCL4000 soft segment, respectively, indicating a smart Materials 2019, 12, 4011 9 of 15 membrane with double temperature switch is acquired. There are also two similar phase transition temperature (crystalline melting peaks) of film A (39.56 • C, 56.00 • C), film B (36.04 • C, 57.32 • C), and film C (35.26 • C, 57.49 • C), respectively. The results demonstrate that the coating of TSPU onto BOPET (film B), the addition of REO (film C) and the incorporation of nanoliposomes containing REOs (film A) remain the double-switch temperature-sensitive characteristic of TSPU film obtained in this study. In addition, compared with the acquired TSPU, the lower phase transition temperature of film A increases slightly from 35.26 to 39.56 • C. This could be attributed to the enhanced rigidity and limited motion of PEG2000 soft segment resulting from the interaction between nanoliposomes and PEG2000 soft segment.  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 temperature sensitive property of PEG2000 and PCL4000 soft segment, respectively, indicating a smart membrane with double temperature switch is acquired. There are also two similar phase transition temperature (crystalline melting peaks) of film A (39.56 °C, 56.00 °C), film B (36.04 °C, 57.32 °C), and film C (35.26 °C, 57.49 °C), respectively. The results demonstrate that the coating of TSPU onto BOPET (film B), the addition of REO (film C) and the incorporation of nanoliposomes containing REOs (film A) remain the double-switch temperature-sensitive characteristic of TSPU film obtained in this study. In addition, compared with the acquired TSPU, the lower phase transition temperature of film A increases slightly from 35.26 to 39.56 °C. This could be attributed to the enhanced rigidity and limited motion of PEG2000 soft segment resulting from the interaction between nanoliposomes and PEG2000 soft segment. XRD patterns of film A, B, C, TSPU and BOPET are depicted in Figure 4b. XRD pattern of TSPU has peaks of 2θ mainly at 21.6°, 22.2 and 23.9° which correspond to the crystalline peaks of the soft segment and the hard segment [29], indicating that the hard segment MDI does not destroy the crystalline morphology of the soft segment PEG and PCL. There are also two similar dominant characteristic peaks 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 imply that coating of TSPU onto BOPET (film B), the addition of REO (film C) and the incorporation of nanoliposomes containing REOs (film A) does not disrupt the intrinsic crystalline morphology of TSPU film obtained in this study. This is in accordance with DSC results that the TSPU based complex films still have temperature response characteristics. Furthermore, compared with pure TSPU film, the peak intensity of film A, B and C all decrease apparently, demonstrating a decrease of crystallinity of the soft segment or hard segment. Besides, all the BOPET based films have similar peak of 2θ at 26.0° (film A), 26.0° (film B) and 26.1° (film C) compared with BOPET (26.9°), implying that film A, B and C remain the crystalline morphology of pure BOPET film. Moreover, with increase of peak intensity, the crystallinity of film A, B and C increase compared with pure BOPET film.

Double Switch Temperature Sensitive Characterization
Porosity ε and WVTR of TSPU and PU films at different temperatures ranging from 15 °C to 65 °C are described in Figure 5a,b, respectively. The diagram shows that both porosity and WVTR of the control film PU without temperature sensibility rise slightly as regular with the increase of temperature. However, the porosity and WVTR of TSPU film undergo two irregular significant increase around phase transition temperature of 35. 26    XRD patterns of film A, B, C, TSPU and BOPET are depicted in Figure 4b. XRD pattern of TSPU has peaks of 2θ mainly at 21.6 • , 22.2 and 23.9 • which correspond to the crystalline peaks of the soft segment and the hard segment [29], indicating that the hard segment MDI does not destroy the crystalline morphology of the soft segment PEG and PCL. There are also two similar dominant characteristic peaks 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 imply that coating of TSPU onto BOPET (film B), the addition of REO (film C) and the incorporation of nanoliposomes containing REOs (film A) does not disrupt the intrinsic crystalline morphology of TSPU film obtained in this study. This is in accordance with DSC results that the TSPU based complex films still have temperature response characteristics. Furthermore, compared with pure TSPU film, the peak intensity of film A, B and C all decrease apparently, demonstrating a decrease of crystallinity of the soft segment or hard segment. Besides, all the BOPET based films have similar peak of 2θ at 26.0 • (film A), 26.0 • (film B) and 26.1 • (film C) compared with BOPET (26.9 • ), implying that film A, B and C remain the crystalline morphology of pure BOPET film. Moreover, with increase of peak intensity, the crystallinity of film A, B and C increase compared with pure BOPET film.
Porosity ε and WVTR of TSPU and PU films at different temperatures ranging from 15 • C to 65 • C are described in Figure 5a,b, respectively. The diagram shows that both porosity and WVTR of the control film PU without temperature sensibility rise slightly as regular with the increase of temperature. However, the porosity and WVTR of TSPU film undergo two irregular significant increase around phase transition temperature of 35.26 • C and 56.98 • C. Specifically, the porosity of TSPU increase from 19.81% at 25 • C to 33.67% at 35 • C and from 48.49% at 55 • C to 68.24% at 65 • C, showing two significant improvements as high as 170% and 141%, respectively. Consistent with the porosity increase tendency, the WVTR of TSPU increase from 70.49 g/m 2 ·24 h at 25 • C to 145.71 g/m 2 ·24 h at 35 • C and from 306.32 g/m 2 ·24 h at 55 • C to 578.54 g/m 2 ·24 h at 65 • C, showing two significant improvements as high as 207% and 189%, respectively. The results indicate that with the phase transition of PEG2000 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 case, WVTR of TSPU determined by porosity illustrates that the TSPU film fabricated in this study has an intelligent temperature response characteristic with a double switch. The results indicate that with the phase transition of PEG2000 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 case, WVTR of TSPU determined by porosity illustrates that the TSPU film fabricated in this study has an intelligent temperature response characteristic with a double switch.  There is no characteristic absorption peak of -NCO in the range of 2260-2280 cm −1 , while an absorption peak appears at 1725 cm −1 , indicating that carbamate group (-NHCOO-) has been produced through the nucleophilic reaction between -NCO and -OH [8]. The incorporation of REO loaded lyophilized nanoliposomes into TSPU does not alter most of the characteristic peak of TSPU except minus shifts of 3311 to 3313 cm −1 , 1305 to 1307 cm −1 , 1237 to 1227 cm −1 , and 952 to 950 cm −1 , respectively. This reveals that no chemical bond is newly built up between REO loaded lyophilized nanoliposomes and TSPU, while between which exists a weak physical interaction. In the contrast, the direct incorporation of REO into TSPU dramatically changes some of the characteristic peaks of TSPU. The peak at 3311 cm −1 not only shifts to 3323 cm −1 , but also substantially decreases the intensity of peak. The peaks at 1305, 1019 and 952 cm −1 also have a relatively large scaled shift to 1294, 1046 and 960 cm −1 . The results indicate that the physical interaction between REO and TSPU is stronger than which between lyophilized nanoliposomes encapsulating REO and TSPU.

Characterization of Controlled Release Complex Film
SEM pictures performing surface micromorphology of TSPU side and cross-section of film A, B and C are exhibited in Figure 6. It can be observed from the TSPU side surface images that film B as blank control film is relatively dense and smooth, while film A incorporated with nanoliposomes containing REOs is slightly uneven with aggregated nanoliposomes and small amount of micropores, and film C as REO control film has more micropores compared with film A. The micropores might be formed by the volatilization of REO during the film drying process. Moreover, the less micropores of film A compared with film C is probably due to the decline of REO volatilization, resulting from the reinforced interaction between the nanoliposomes and the soft segment in TSPU film in agreement with DSC results in this study (Section 3.2). Likewise, the amount of micropores presented  There is no characteristic absorption peak of -NCO in the range of 2260-2280 cm −1 , while an absorption peak appears at 1725 cm −1 , indicating that carbamate group (-NHCOO-) has been produced through the nucleophilic reaction between -NCO and -OH [8]. The incorporation of REO loaded lyophilized nanoliposomes into TSPU does not alter most of the characteristic peak of TSPU except minus shifts of 3311 to 3313 cm −1 , 1305 to 1307 cm −1 , 1237 to 1227 cm −1 , and 952 to 950 cm −1 , respectively. This reveals that no chemical bond is newly built up between REO loaded lyophilized nanoliposomes and TSPU, while between which exists a weak physical interaction. In the contrast, the direct incorporation of REO into TSPU dramatically changes some of the characteristic peaks of TSPU. The peak at 3311 cm −1 not only shifts to 3323 cm −1 , but also substantially decreases the intensity of peak. The peaks at 1305, 1019 and 952 cm −1 also have a relatively large scaled shift to 1294, 1046 and 960 cm −1 . The results indicate that the physical interaction between REO and TSPU is stronger than which between lyophilized nanoliposomes encapsulating REO and TSPU.

Characterization of Controlled Release Complex Film
SEM pictures performing surface micromorphology of TSPU side and cross-section of film A, B and C are exhibited in Figure 6. It can be observed from the TSPU side surface images that film B as blank control film is relatively dense and smooth, while film A incorporated with nanoliposomes containing REOs is slightly uneven with aggregated nanoliposomes and small amount of micropores, and film C as REO control film has more micropores compared with film A. The micropores might be formed by the volatilization of REO during the film drying process. Moreover, the less micropores of film A compared with film C is probably due to the decline of REO volatilization, resulting from the reinforced interaction between the nanoliposomes and the soft segment in TSPU film in agreement with DSC results in this study (Section 3.2). Likewise, the amount of micropores presented in the 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.
in the 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. Both the test data and fitting curves of REO releasing proportion out of film C and A at 25 °C, 40 °C and 60 °C are depicted in Figure 7a,b, respectively. The goodness of fit is evaluated by means of the root mean square error (RMSE) listed in Table 2. The results show that the model satisfactorily fits the test data, suggesting the adopted model is suitable for characterization of REO release from the complex film. The calculated Ds are presented in Table 2, and the increase tendency of D with temperature ascending is described in Figure 7c. Both the test data and fitting curves of REO releasing proportion out of film C and A at 25 • C, 40 • C and 60 • C are depicted in Figure 7a,b, respectively. The goodness of fit is evaluated by means of the root mean square error (RMSE) listed in Table 2. The results show that the model satisfactorily fits the test data, suggesting the adopted model is suitable for characterization of REO release from the complex film. The calculated Ds are presented in Table 2, and the increase tendency of D with temperature ascending is described in Figure 7c. in the 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. Both the test data and fitting curves of REO releasing proportion out of film C and A at 25 °C, 40 °C and 60 °C are depicted in Figure 7a,b, respectively. The goodness of fit is evaluated by means of the root mean square error (RMSE) listed in Table 2. The results show that the model satisfactorily fits the test data, suggesting the adopted model is suitable for characterization of REO release from the complex film. The calculated Ds are presented in Table 2, and the increase tendency of D with temperature ascending is described in Figure 7c.   It can be observed that the release of REO from both films (film C and A) at all temperatures (25 °C, 40 °C and 60 °C) has not reached equilibrium in 60 days, and the mass rate of released REO was less than 10%, indicating a very slow release of REO. This is in accordance with the low value of D at magnitude of 10 −14 (cm 2 ·s −1 ). Moreover, Ds of film A are smaller than that of film C at each temperature tested, implying a significant barrier and protection effect of nanoliposome layer in this study. Although Ds of film C are larger than Ds of film A at each temperature tested, the relatively low release rate of REO represented by D at magnitude of 10 −14 (cm 2 ·s −1 ) is probably caused by the strong interaction between directly added REO and TSPU as discussed before according to FT-IR results. Furthermore, with the temperature ascending, D of both film C and A undergoes an abnormal dramatically increase (Figure 7c) against Arrhenius theory, in which D is exponentially related to temperature. This demonstrates that both complex film C and A have remained the double 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 enlarging the free volume hole size and accelerating micro-Brownian motion of soft segment in TSPU.
The results of the DPPH radical scavenging test for film A, B and C are shown in Table 3. It can be concluded from Table 3 that the DPPH radical scavenging activity of film A gradually increases during the period of 1440 h (60 d). The extremely low DPPHs of film B might be caused by experimental error. For film C, the DPPH free radical scavenging activity is higher within 720 h (30 d) whereas lower at 1440 h (60 d) compared with film A. The results demonstrate that film A has a lasting antioxidant effect due to the controlled release property of nanoliposomes. Besides, the obtained multicomponent film can be recycled by solving isolation method according to research of Patrizia et al. [31]   It can be observed that the release of REO from both films (film C and A) at all temperatures (25 • C, 40 • C and 60 • C) has not reached equilibrium in 60 days, and the mass rate of released REO was less than 10%, indicating a very slow release of REO. This is in accordance with the low value of D at magnitude of 10 −14 (cm 2 ·s −1 ). Moreover, Ds of film A are smaller than that of film C at each temperature tested, implying a significant barrier and protection effect of nanoliposome layer in this study. Although Ds of film C are larger than Ds of film A at each temperature tested, the relatively low release rate of REO represented by D at magnitude of 10 −14 (cm 2 ·s −1 ) is probably caused by the strong interaction between directly added REO and TSPU as discussed before according to FT-IR results. Furthermore, with the temperature ascending, D of both film C and A undergoes an abnormal dramatically increase (Figure 7c) against Arrhenius theory, in which D is exponentially related to temperature. This demonstrates that both complex film C and A have remained the double 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 enlarging the free volume hole size and accelerating micro-Brownian motion of soft segment in TSPU.
The results of the DPPH radical scavenging test for film A, B and C are shown in Table 3. It can be concluded from Table 3 that the DPPH radical scavenging activity of film A gradually increases during the period of 1440 h (60 d). The extremely low DPPHs of film B might be caused by experimental error. For film C, the DPPH free radical scavenging activity is higher within 720 h (30 d) whereas lower at 1440 h (60 d) compared with film A. The results demonstrate that film A has a lasting antioxidant effect due to the controlled release property of nanoliposomes. Besides, the obtained multicomponent film can be recycled by solving isolation method according to research of Patrizia et al. [31]. 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.

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.