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Food Chemistry

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Gallic acid liposomes decorated with lactoferrin: Characterization, in vitrodigestion and antibacterial activityYating Zhang1, Chuanfen Pu1, Wenting Tang⁎, Shiqing Wang⁎, Qingjie SunSchool of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China

A R T I C L E I N F O

Keywords:Gallic acidLiposomeLactoferrinAntibacterial activity

A B S T R A C T

Gallic acid liposomes (GA-LIP) and gallic acid liposomes decorated with lactoferrin (LF-GA-LIP) were fabricated,and their physiochemical, in vitro digestion and antibacterial activity were analysed. The average particle size ofLF-GA-LIP was larger than that of GA-LIP, and the former had a higher encapsulation efficiency and storagestability. Electrostatic interaction existed between lactoferrin and the phospholipid bilayer. Spherical structuresof both were observed by transmission electron microscopy and atomic force microscopy. Gallic acid existed inan amorphous form in both liposomes. Hydrogen bond was formed between the hydroxyl groups of gallic acidand the polar head of phospholipid in the liposomes. LF-GA-LIP displayed a delayed-release effect comparedwith GA-LIP in simulated digestion. It exerted higher antibacterial properties against Escherichia coli andStaphylococcus aureus than GA-LIP. The findings suggested that the liposomes decorated with lactoferrin could bedeveloped as a favourable delivery system for a potential application in the food industry.

1. Introduction

Gallic acid (3,4,5-trihydroxybenzoic acid) is a polyphenolic com-pound commonly found in nature. It has a wide range of applications infood, medicine, and chemical industries because of its antioxidant,antitumor, and other properties. However, its unpleasant taste andsensitivity to oxygen, light and extreme temperatures gives rise to aseries of difficulties in potential applications (Celli, Ghanem, andBrooks, 2015). The low half-life and rapid clearance by the body alsolimits its use as a nutraceutical (Shahrzad, Aoyagi, Winter, Koyama, &Bitsch, 2001). Thus, there is a necessity to develop a formulation thatcan exert a protective effect in processing and storage, and maintain itsconcentration in the body delivery over a longer period of time. En-capsulation provides an effective means to protect it from unfavourableenvironmental factors and to increase the bioavailability when takenorally (Rosa et al., 2013).

Liposomes are a colloidal delivery system that can self-assemble.They were formulated by dispersing a polar lipid in an aqueousmedium. Liposomes have a hydrophilic core and one or more

phospholipid bilayer membranes, which can encapsulate both hydro-philic and lipophilic substances. They have many advantages that havebeen shown to improve the bioavailability, stability, sustained releaseand cellular antioxidant activity of the encapsulated nutraceuticalcompounds (Chen et al., 2015; Ding et al., 2011; Pu, Tang, Li, Li, & Sun,2019). Thus, liposome encapsulation might be a promising approach toimprove the stability and bioavailability of gallic acid. However, lipo-somes tend to aggregate, fuse or degrade due to its soft material attri-butes (Rahnfeld, Thamm, Steiniger, Van, and Luciani, 2018). The un-stable phenomena likely to occur during storage, especially in thecolloidal dispersions, resulting from the interaction between liposomalparticles and the effects of the external environment factors. Thesedefects cause morphological, structural changes and ultimately leakageof the encapsulated compounds, which greatly limit their application(Mortazavi, Mohammadabadi, Khosravi-Darani, & Mozafari, 2007).Recently, it has been proposed that liposomes can be surface modifiedby polysaccharides or proteins to rigidify the structure and improvetheir stability (Liu, Ye, Han, & Han, 2018).

Lactoferrin is an important non-haem iron-binding glycoprotein in

https://doi.org/10.1016/j.foodchem.2019.04.116Received 22 November 2018; Received in revised form 28 March 2019; Accepted 30 April 2019

Abbreviations: GA-LIP, gallic acid liposome; LF-GA-LIP, gallic acid liposome decorated with lactoferrin; PDI, polydispersity index; FTIR, Fourier transform infraredspectroscopy; XRD, X-ray diffraction; TEM, transmission electron microscopy; AFM, atomic force microscopy; CLSM, laser scanning confocal microscopy; AO,acridine orange; PI, propidium iodide; EE, encapsulation efficiency; DSC, differential scanning calorimetry; MIC, minimum inhibitory concentration; DIZ, diameter ofinhibition zone; DLS, dynamic light scattering; SSF, simulated saliva fluid; SGF, simulated gastric fluid; SIF, simulated intestine fluid; BHT, butylated hydroxytoluene;FFA, free fatty acid; PBS, phosphate-buffered saline; LB, lysogeny broth

⁎ Corresponding author.E-mail addresses: twtfly@126.com (W. Tang), 1114254307@qq.com (S. Wang).

1 Yating Zhang and Chuanfen Pu contributed equally to this work.

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milk. It is mainly expressed and secreted by breast epithelial cells.Lactoferrin not only participates in the transportation of iron but alsohas antioxidant, anticancer, immune regulation, and other biologicalfunctions (Tonguc-Altin et al., 2015). Lactoferrin has been used tomodify procationic liposomes to act as brain specific targeting ligands.The system was characterized by an enhanced accumulation in brain invivo (Chen et al., 2010). The cellular delivery efficiency has been im-proved and the growth of cancer cells has been inhibited by the surfacemodification of doxorubicin-loaded liposomes with lactoferrin (Chenet al., 2011). The permeability of the neuron growth factor across theblood–brain barrier can also be increased by encapsulation in lacto-ferrin-decorated liposomes (Kuo & Wang, 2014).

GA has previously been encapsulated in mesoporous silica nano-particles (Iraji, Ganji, & Rashidi, 2018) and poly (ethylene oxide)/zeinnanofibers (Acevedo et al., 2018). Liposomal gallic acid has also beenprepared as antioxidant in different textiles designed to be in contactwith the skin as biofunctional textiles (Martí et al., 2014). However, tothe best of our knowledge, there has been no previous study on gallicacid liposomes modified with lactoferrin (LF-GA-LIP). In the presentstudy, gallic acid liposomes (GA-LIP) and LF-GA-LIP were fabricatedfirst, and their physicochemical properties and stabilities were eval-uated. Then, the release kinetics of gallic acid in both liposomes wasinvestigated by simulated digestion. Finally, the antibacterial activitiesof GA-LIP and LF-GA-LIP against Escherichia coli and Staphylococcusaureus were evaluated. The results might be useful for the developmentof effective delivery systems for gallic acid.

2. Materials and methods

2.1. Materials

Gallic acid (> 98%), mucin (> 99%), pepsin, pancreatin werepurchased from Sigma-Aldrich Co. (Missouri, USA). Lecithin fromsoybean was purchased from Shanghai Yuanju Biological TechnologyCo. (Shanghai, China). Lactoferrin (> 95%) and cholesterol was ob-tained from Yuanye Biotechnology Co. (Shanghai, China). E. coli ATCC33456 and S. aureus ATCC 25,923 were obtained from GuangdongHuankai Microbial Sci. & Tech. Co, Ltd (Guangzhou, China). All otherreagents were of analytical grade.

2.2. Preparation of GA-LIP and LF-GA-LIP

GA-LIP were prepared by a thin-layer dispersion method, as de-scribed in Liu’s previous study (Liu et al., 2011). Phospholipid, cho-lesterol, Tween-80, and vitamin E (in a mass ratio of 6:1:1.8:0.12) weredissolved in ethanol and then evaporated into a thin film using a rotaryevaporator under 40 °C. The dried lipid film was rehydrated withphosphate-buffered saline (PBS, 0.05M, pH7.0). The concentration oflipids (phospholipid and cholesterol) was 1mg/ml. The liposomal sus-pension was then subjected to a probing sonication process in an icebath for 15min at 160W with a sequence of 1 s of sonication and 1 s ofrest (Sonics & Materials, Inc., 20 kHz). Liposomes loaded with 2%, 4%,6%, and 8% gallic acid (designated as 2% GA-LIP, 4% GA-LIP, 6% GA-LIP, and 8% GA-LIP) were further fabricated by hydrating the lipid filmwith PBS containing gallic acid. The unencapsulated gallic acid wasseparated by ultracentrifugation (Fritze, Hens, Kimpfler, Schubert, &Peschka-Süss, 2006). To prepare the LF-GA-LIP samples, the lactoferrinsolution, the pH of which adjusted to 9.0, was added dropwise to theliposome suspension with the same pH by magnetic stirring at a speedof 100 rpm. The volume ratio of the liposome suspension to the lacto-ferrin solution was 1:1. The final concentration of lactoferrin was 2mg/ml. Bare liposomes, which refer to the vesicles without lactoferrindecoration and gallic acid encapsulation were also prepared as thecontrol.

2.3. Physicochemical characterization of liposomes

2.3.1. Measurements of particle size and ζ-potentialThe average particle size, polydispersity index (PDI), and ζ-potential

of the liposomes were measured by dynamic light scattering (DLS)using a Zetasizer (Nano-ZS, Malvern Instruments, Worcestershire, UK).The sample was diluted 10 times prior to measurement with PBS(0.05M, pH7.0) to avoid multiple scattering effects. All measurementswere performed at 25 °C, and each sample was analyzed in triplicate.

2.3.2. Encapsulation efficiency (EE)The EE of GA-LIP and LF-GA-LIP were determined using an ultra-

centrifugation technique according to the method of Fritze et al.(2006). Ultracentrifugation was performed (130000× g, 3 h, 4 °C) toseparate the unloaded gallic acid. The liposome pellet was redispersedin PBS. The amount of encapsulated gallic acid was determined at280 nm after lysis of the liposomes with Triton X-100 (final con-centration 0.5% v/v). The EE of gallic acid was then calculated ac-cording to the following equation:

=EE% W /W 100%2 1

where W1 is the total gallic acid weight added in liposome preparation,mg; W2 is the weight of encapsulated gallic acid, mg.

2.3.3. Storage stability evaluationThe liposome suspensions were stored at 4 °C in the dark for

20 days, and the samples were taken every 5 days. The particle size,PDI, ζ-potential and retention rate of gallic acid of the samples weredetermined.

2.4. Morphology observation

The morphologies of the liposomes were analyzed by transmissionelectron microscopy (TEM) and atomic force microscopy (AFM). Thefreshly prepared liposomes were diluted to a concentration 0.5mg/mlwith PBS (pH 7.0). A drop of the diluted dispersion was then placed ona carbon film grid and negatively dyed with phosphotungstic acid andthen subjected to TEM (JEM-2200FS, JEOL Ltd., Japan). For AFM, 1 μlof the sample was pipetted onto the surface of the newly dissociatedmica and allowed to spread for 30min at room temperature. The micasurface was then gently washed with ultrapure water to remove anysalts and unabsorbed particles. After sufficient drying, the sample wasplaced under a scanning probe (Si probe) of an AFM and scanned inScan Asyst mode (frequency: 320 kHz, spring coefficient: 42 N/m).

2.5. Fourier transform infrared spectroscopy (FTIR)

Sample spectra were recorded using a Fourier-transform infraredspectrometer (Vertex 70v, Bruker, Germany). The sample was droppedon a blank KBr sheet and baked under an infrared light before scanning.The scanning range was 400–4000 cm−1, the resolution was set to4 cm−1.

2.6. X-ray diffraction (XRD) analysis

The crystal structures of the samples were analyzed by X-ray dif-fraction (AXS D8 ADVANCE; Bruker, Germany) using the method re-ported by (Ge et al., 2018). The scanning range (2θ) was from 10° to60°, and the step size was 0.01°.

2.7. Differential scanning calorimetry (DSC) analysis

The DSC analyses were performed on a DSC-Q1000 differentialscanning calorimetry (TA Instrument, USA) at a scanning speed of 10 C/min. Samples (7.0 mg) were heated from 25 to 300 °C under nitrogenGas flow. The measurements were carried out in triplicate.

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2.8. In vitro digestion

To investigate the in vitro digestion of gallic acid, a simulated modelconsisting of the mouth, stomach, and intestine was used as previouslydescribed (Li et al., 2018). All solutions and samples were pre-in-cubated with shaking at 37 °C and maintained at this temperaturethroughout the digestion.

2.8.1. Mouth phaseAccording to previous studies, the in vitro digestion process begins

in the oral cavity; we prepared simulated saliva fluid (SSF) using theconcentrations and types of various salts described in the method ofMao and McClements (Mao & Mcclements, 2012). In the digestion, 2mlof liposome suspension was placed in a beaker and mixed with 2ml ofSSF, and then the pH of the mixture was adjusted to 6.8 and incubatedfor 10min in a 100 rpm shaker.

2.8.2. Gastric phaseA 1.5-ml sample was mixed with 13.5ml of physiological saline

(containing 140mmol/l NaCl, 5 mmol/l KCl, and 150mmol/l butylatedhydroxytoluene (BHT)) and stirred for 10min. The pH of the solutionwas adjusted to 2.0 with 0.1mol/l HCl, and then 1ml of pepsin at a

concentration of 3.2 g/l was added to simulate gastric juice (Liang,Shoemaker, Yang, Zhong, & Huang, 2013).

2.8.3. Intestine phaseThe pH of the system was adjusted to 7.5 with 0.1mol/l NaOH, and

4.5 ml pancreatin was added to simulate intestinal fluid (Zhao et al.,2011). Within 2 h of digestion, 0.1 mol/l of NaOH solution was added tomaintain the pH of the system at 7.5, and the amount of NaOH con-sumed during the experiment was recorded by automatic acid-base ti-trator to calculate the amount of fatty acid released.

2.8.4. Analysis of the release characteristics of gallic acidThe released gallic acid in the digestive fluid was separated by

centrifugation (130000×g, 3 h) and then determined at 280 nm. Toevaluate the release characteristics of gallic acid, the classical powerexponential equation found in the Korsmeyr-Peppas Model was used todescribe the release mechanism of the carrier system (Zhao et al.,2011). This model has been shown to reflect the release mechanism ofliposomes (Yan, Barnes, Fornasiero, & Prestidge, 2009). The formulawas as follows:

=MM

ktt n

Fig. 1. (a) TEM images of GA-LIP and LF-GA-LIP and (b) AFM images of GA-LIP and LF-GA-LIP.

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Mtwas the released amount at time t, mg; M was the releaseamount at time , mg; Mt/M is the release percentage at time t, %; k isthe release rate constant; the release index n characterizes the char-acteristic parameters of the release mechanism. For the globular model,n < 0.43 refers to Fickian diffusion, irregular diffusion (smoothing anddiffusion) was between 0.43 and 0.85 and n > 0.85 corresponds tocase-II transport (Yan et al., 2009).

2.8.5. Determination of free fatty acid (FFA) releaseThe amount of released FFA was determined based on the volume of

NaOH consumed in the simulated intestinal digestion:

= × × × ×V C Mm

FFA release(%) 10 100%1 1 13

1

where V1 is the volume of NaOH solution consumed, ml; C1 is theconcentration of NaOH solution used during titration, mmol/ml; M1 isthe average molecular weight of phosphatidylcholine, g/mol; and m1 isthe total mass of phosphatidylcholine, g.

2.9. Determination of antibacterial properties

2.9.1. Determination of minimum inhibitory concentration (MIC)E. coli and S. aureus were used as the model microorganisms to test

the antibacterial activities. Briefly, both microorganisms were collectedat their midlogarithmic phase and resuspended in PBS (pH 7.2, 10mM)to obtain a final microbial density of about 106 CFU/ml. Then, 50 μl ofgradiently diluted liposome samples was placed in sterile flat-bottomed96-well plates, after which 100 μl of the cell suspension and 100 μl of LB(lysogeny broth) media were added. The samples were then incubatedat 37 °C for 18 h. The absorbance of each well at 630 nm was measured.PBS (pH 7.2, 10mM) solution was used as the negative control. Gallicacid and lactoferrin were also evaluated for comparison. The minimumconcentration of the sample at which OD630 (by Bio-Rad LaboratoriesLnC, iMarK) no longer increased compared to the initial value was theminimum inhibitory concentration (MIC) of the sample.

2.9.2. Determination of diameter of inhibition zone (DIZ)The DIZ size was determined using the filter paper diffusion method

described by Li et al (Li et al., 2014). The filter paper was punched intoa 6-mm diameter disc and sterilized. The sterile filter paper discs wererespectively impregnated with the samples (1 MIC), and placed on thesurface of the culture media seeded with the test microorganisms. Discsimpregnated with sterilized PBS (pH 7.2, 10mM) were used as negativecontrol and the discs with gallic acid and lactoferrin (1 MIC) were alsoevaluated. After standing for 5min, the culture dish was incubated at37 °C for 24 h and then the diameters of the inhibition zone weremeasured. Each experiment was repeated at three times and mean ofthe diameter of inhibition zones was calculated.

2.9.3. Laser scanning confocal microscopy (LSCM)The treated bacterial suspension was double stained with acridine

orange (AO)/propidium iodide (PI) for LSCM observation. Briefly, thetreated bacterial suspension was washed twice with PBS (pH 7.2,10mM). Then, 10 μl of AO (50 μg/ml) and 10 μl of PI (50 μg/ml) weresequentially added to the resuspended cells (106 CFU/ml). The mixturewas washed in PBS after incubating in the dark at 4 °C for 30min. Thecells were observed by a laser scanning confocal microscope (LEICATCS SP5 II FM, Leica Microsystems, Wetzlar, Germany).

2.10. Statistical analysis

All the experiments were repeated at least three times, the resultsare expressed as means ± standard deviations (SD). Data were sub-jected to statistical analysis using SPSS software (version 19.0).Statistical test were performed by t-test and differences with p < 0.05was considered statistically significant.

Fig. 2. (a) FTIR of lactoferrin, LF-GA-LIP, GA-LIP and bare liposome; (b) X-rayof GA-LIP, gallic acid, lactoferrin and LF-GA-LIP and (c) DSC of gallic acid,lactoferrin, GA-LIP and LF-GA-LIP.

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3. Results and discussion

3.1. Characterization of GA-LIP and LF-GA-LIP

GA-LIP loaded with different concentrations (2%–8%) of gallic acidwere fabricated and evaluated first to obtain the optimal loadingamount. The average size of freshly prepared bare liposomes withoutgallic acid was 98.2 ± 1.3 nm with a negative charge of−33.3 ± 1.7mV (Fig. S1). The PDI value of 0.152 ± 0.004 indicatedthat the system had uniform dispersibility. The average particle sizewas increased by the loading of gallic acid up to 6%. The interaction ofgallic acid with the polar headgroup of phospholipid in the bilayer ledto its spanning orientation in the liposomal membrane, which couldexpand the liposome volume and size (Singh, Mohan, Semalty, &Semalty, 2011). However, further increase of the loading amount ofgallic acid (8%) did not result in the larger particle size. This phe-nomenon might be due to the saturation of gallic acid in the vesicles(Huang, Su, Zheng, & Tan, 2017), which was also supported by the factthat the EEs of GA-LIP decreased significantly when the loading amountincreased from 4% to 8%. Although the loading of gallic acid increasedthe polydispersity, the PDI values of all samples were still less than 0.3,indicating a relatively homogenous size distribution. The absolute va-lues of the ζ- potential of all samples were greater than 30mV. Thestrong electrostatic repulsion existed between the negatively chargedparticles, maintaining the particles set apart from each other. To sumup, GA-LIP loaded with 2% gallic acid had higher EE and lower particlesize. Therefore, it was chosen to carry out the subsequent experiment.

The charge states of lactoferrin and GA-LIP affect the feasibility ofdecoration of lactoferrin on the surface of the liposomes. Therefore, theζ-potentials of lactoferrin and GA-LIP under different pH conditionswere investigated. As shown in Fig. S2, the ζ-potential of lactoferrinincreased from −13.4 ± 0.72mV to 7.83 ± 0.47mV when the pHvalue increased from 3.0 to 10.0; the net charge was zero at pH 8.34.The ζ- potential of GA-LIP varied from −37.9 ± 0.57mV to−24.5 ± 0.34mV in the pH range of 3.0–10.0. At pH 9.0, positivelycharged lactoferrin could interact with the negatively charged GA-LIPvia electrostatic interaction. As a result, pH 9.0 was selected to fabricateLF-GA-LIP.

DLS revealed that the freshly prepared LF-GA-LIP loaded with 2%gallic acid showed an average particle size of 153.2 ± 1.4 nm with aPDI of 0.252 ± 0.002. The larger size compared with GA-LIP might bedue to the surface coating of lactoferrin on the outside of the liposome.The ζ- potential of LF-GA-LIP at pH 9.0 was higher than that of GA-LIP,which might be attributed to the positive charge of lactoferrin. The EEof LF-GA-LIP (97.3 ± 2.5%) was greater than that of GA-LIP(95.2 ± 3.6%). The morphologies of the fabricated GA-LIP and LF-GA-LIP were observed by TEM and AFM (Fig. 1). All methods showed thatGA-LIP and LF-GA-LIP were almost spherical, and the vesicles wereevenly dispersed without obvious aggregation and fusion. The TEManalysis results of LF-GA-LIP also showed uniformity and dispersionwhich proved that the modification of lactoferrin did not affect thesurface morphology of the liposome. In addition, the particle sizes ofGA-LIP (90.3 ± 3.4 nm) and LF-GA-LIP (117.3 ± 2.8 nm) assessedusing TEM were all smaller than those measured by DLS in Fig. S3 (GA-LIP, 100.4 ± 1.2 nm; LF-GA-LIP, 153.2 ± 1.4 nm). The reason for this

Table 1Particle size, ζ-potential and PDI of GA-LIP and LF-GA-LIP during in vitro digestion.

LF-GA-LIP GA-LIP

Oral Stomach Intestine Oral Stomach Intestine

particle size(nm) 218.3 ± 3.4a 243.5 ± 4.5b 486.4 ± 5.2c 207.3 ± 3.4a 256.5 ± 5.3b 467.4 ± 4.7c

PDI 0.653 ± 0.012a 0.542 ± 0.021b 0.521 ± 0.018b 0.777 ± 0.12a 0.437 ± 0.017b 0.432 ± 0.032b

ζ-potential(mv) −31.40 ± 2.14a −18.41 ± 1.37b −18.72 ± 2.56b −27.60 ± 1.23a −10.44 ± 0.89c −18.7 ± 1.32b

Results are expressed as mean value ± standard deviation (n=3). Different letters in the same row represent a significant difference (P < 0.05).

Fig. 3. (a) Gallic acid leakage rate and (b) FFA release of GA-LIP and LF-GA-LIP.

Table 2Diameter of the inhibition zone (DIZ) and minimum inhibitory concentration(MIC) of different treatments against E. coli and S. aureus.

Strain treatment MIC/μg/ml DIZ/mm

E. coli LF-GA-LIP 0.25 12.0 ± 1.2d

GA-LIP 0.25 10.1 ± 0.9d

Gallic acid 1 6.8 ± 0.1b

Lactoferrin 1 6.3 ± 0.1b

S. aureus LF-GA-LIP 0.25 10.2 ± 2.3c

GA-LIP 0.25 8.6 ± 1.3c

Gallic acid 1 7.1 ± 0.2b

Lactoferrin 1 6.5 ± 0.1b

Results are expressed as mean value ± standard deviation (n= 3). Differentlower letters in the same column represent a significant difference (P < 0.05).

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might be due to that the hydrodynamic diameter was evaluated in DLS(Tan et al., 2015). While for TEM, the vesicles were dried on the carbonfilm grid and determined without the hydrated portion (Liu & Park,2010). In addition, the particle sizes of GA-LIP and LF-GA-LIP deducedfrom the AFM images were also a bit different from the data determinedusing DLS. Some vesicles undergo morphological changes during AFMsample preparation (Pentak, 2014). The average heights of GA-LIP andLF-GA-LIP liposomes in AFM were smaller than their diameters due tothe fact that the liposomes collapsed on the mica (Richter, Bérat, &Brisson, 2006).

3.2. Comparison of stability between GA-LIP and LF-GA-LIP during storage

The stability comparison between LF-GA-LIP and GA-LIP duringstorage is shown in Fig. S3. Overall, the particles size of both liposomesincreased with the time during the storage period at 4 °C. The particlesize of LF-GA-LIP was larger than that of GA-LIP at the same time point,which proved the existence of lactoferrin around the surface of the li-posomes. Although the PDI values of both fluctuated with time, theywere all still lower than 0.3, which proved that both samples showed a

narrow size distribution up to 20 days. The absolute value of the ζ-potential of LF-GA-LIP and GA-LIP decreased with the increase in sto-rage time, suggesting the electrostatic repulsion became lower and theaggregation trend increased. The aggregation and hydrolysis of phos-pholipids during storage led to the disruption of the lipid bilayerstructure and ultimate leak of gallic acid. It should be noted that LF-GA-LIP showed a higher retention rate of gallic acid than GA-LIP did,suggesting the protect effect of lactoferrin decoration on the structuralintegrity of liposomes during storage.

3.3. FTIR analysis

FTIR was used to investigate the possible interaction between gallicacid, lactoferrin, and phospholipid bilayer (Mady, Darwish, Khalil, &Khalil, 2009). The bare liposomes showed an adsorption band at2920 cm−1, which reflected the symmetric vibration of –CH2 and –CH3

of the phospholipid (Fig. 2a). A peak at 1624 cm−1 was due to a C]Obond in the phospholipid. The obtained spectrum of bare liposomes wassimilar to that obtained in previous investigations (Tai et al., 2018).Compared with bare liposomes, GA-LIP exhibited an absorption band

Fig. 4. (a) CLSM images of E. coli grown under different conditions; (b) CLSM images of S. aureus grown under different conditions; (c) TEM images of E. coli grownunder different conditions and (d) TEM images of S. aureus grown under different conditions.

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corresponding to benzene ring C]C stretching vibration at 1521 cm−1,which was a characteristic peak of gallic acid, indicating the existenceof gallic acid in GA-LIP. The position of the C]O bond of phospholipidshifted to 1653 cm−1 in gallic acid loaded liposomes, illustrating thehydrogen bonding between the hydroxyl group of gallic acid and theC]O of the phospholipid (Hincha & Hagemann, 2004). Lactoferrinshowed a characteristic peak at 1090 cm−1, which was generated bythe stretching vibration of the amide group. The absorption peak at1535 cm−1 was the CeN stretching vibration of the amide II band(Pawlikowska-Pawlęga et al., 2013). These characteristic absorptionpeaks still appeared in LF-GA-LIP, suggesting the existence of lacto-ferrin on the surface of GA-LIP.

3.4. The crystallinity state of gallic acid in liposomes

XRD analysis was performed to investigate the crystallinity state ofgallic acid in liposomes as reducing crystallinity has been reported to befavourable for its bioavailability in the gastrointestinal tract. Thecharacteristic peaks of unencapsulated gallic acid confirmed its crys-talline structure (Fig. 2b). However, these peaks disappeared in theXRD patterns of the GA-LIP and LF-GA-LIP, indicating that gallic acidexisted in an amorphous state after encapsulated in the liposomes. Theabsence of peaks and the presence of noise in the XRD pattern of lac-toferrin was related to the detection of amorphous phases, which hasalso been reported in the previous research (Gulão, de Souza, da Silva,Coimbra, & Garcia-Rojas, 2014). The unorganized or random molecularstate of lactoferrin still existed after coating of the liposomes, as shownin the XRD pattern of LF-GA-LIP.

DSC analysis was further conducted to prove the amorphous state ofgallic acid after encapsulated in the liposome. In Fig. 2c, gallic acidshowed a sharp endothermic peak corresponding to a melting point of260 °C, which was the evidence of its crystalline form. However, themelting peak of gallic acid was not observed in the thermogram of GA-LIP, indicating the disappear of its crystal structure (Neo et al., 2013),which was consistent with the X-ray diffraction result that gallic acidexisted in an amorphous state in the liposome.

3.5. In vitro digestion

The stabilities of GA-LIP and LF-GA-LIP under simulated digestionconditions were evaluated in terms of particle size, ζ-potential and PDI.The average particle sizes of GA-LIP and LF-GA-LIP after oral digestionwere 207.3 ± 3.4 nm and 218.3 ± 3.4 nm, respectively, which weresignificantly larger than those of the undigested samples (Table 1). Theincrease in particle size may be due to the swelling and aggregation ofthe liposomes in simulated saliva. The bridging action of the salt andthe polyelectrolyte, and the binding behaviour of mucin to the lipo-somes in the saliva might be the reason for the aggregation of the li-posomes (Mao & Mcclements, 2012). However, the gallic acid leakagerate of the sample in the oral digestion stage remained unchanged(Fig. 3a), which indicates that the liposome was not substantially di-gested in the oral cavity. Although lactoferrin and phospholipids couldbe digested under simulated gastrointestinal conditions, the particlesizes of the liposome continued to increase, probably due to the ad-sorption of the components in the digestive fluid and the digestionproducts (Singh & Sarkar, 2011). After digestion in the simulated gas-tric fluid (SGF), the leakage rate of LF-GA-LIP and GA-LIP were 17.6%and 18.1%, respectively. During the simulated intestine digestion, bothliposomes were further hydrolyzed by pancreatin, leading to the dis-ruption of the vesicles and further release of gallic acid. When the si-mulated intestine digestion was finished, the leakage rate of LF-GA-LIPwas lower than that of GA-LIP, suggesting lactoferrin surface decorationcould delay the release of gallic acid from the liposomes during simu-lated digestion.

Fig. 3b shows that the release of FFA increased rapidly in all sampleswithin 40min of SIF digestion, followed by a slow increase; this result

was consistent with the findings of Mohanraj, Barnes, and Prestidge(2010). The process of phospholipid hydrolysis was accompanied by thedestruction and disintegration of the liposome structure, and the corematerial was gradually released from the bilayer. However, the finalrelease rate of FFA was stable around 40%, while the release rate of LF-GA-LIP was slightly lower than that of GA-LIP. The result suggested thatlactoferrin deposited on the surface of the liposome inhibited the de-struction of the phospholipid layer and leakage of gallic acid to a cer-tain extent.

Table S1 shows the release kinetic parameters for GA-LIP and LF-GA-LIP. Both GA-LIP and LF-GA-LIP followed the Korsmeyr-PeppasModel (R2＞0.92). For the globular model, n < 0.43 suggests Fickiandiffusion, n value between 0.43 and 0.85 indicates irregular diffusion(smoothing and diffusion), and n > 0.85 suggests case-II transport.Here the values of n were> 0.85, revealing that gallic acid was pri-marily swelling-controlled (Case-II transport release mechanism) (Yanet al., 2009).

3.6. Antibacterial activities

The MICs of LF-GA-LIP and GA-LIP against E. coli and S. aureus werelower than those of gallic acid and lactoferrin, which indicated higherantibacterial properties were obtained after loading of gallic acid inboth liposomes (Table 2). At the same concentration, for both strains,the DIZs of LF-GA-LIP were larger than those of GA-LIP for both strains,suggesting that the antibacterial properties of LF-GA-LIP were betterthan those of GA-LIP, which might be partly due to the antibacterialactivity of lactoferrin (Mahdi et al., 2018).

AO/PI were used to stain the different treated bacterial suspensionsfor LSCM observation (Fig. 4). AO dye has the membrane permeabilityto penetrate the cell membrane and stain DNA and RNA at an excitationwavelength of 488 nm and an emission wavelength of 515 nm, emittinggreen fluorescence in living cells and yellow fluorescence in apoptoticcells. PI is a DNA-binding dye that has no membrane permeability andtherefore cannot pass through the living cell membrane; it only entersdead cells. The control group of E. coli and S. aureus were all greenish,and a small amount of yellow appeared, indicating that most of the cellswere living cells. Most of the bacteria treated with GA-LIP were red;only a small portion was green, which suggested that the liposometreatment can increase the permeability of the cell membrane, allowingPI to enter into the cell and bind to the DNA through the damaged cellmembrane. Accordingly, GA-LIP can inhibit the growth of S. aureus andE. coli and inactivate them through a cell membrane damage me-chanism. LF GA-LIP treated bacteria showed more dead cells comparedwith GA-LIP treated ones. In accordance with the result of DIZs, gallicacid and lactoferrin exhibited weaker antibacterial activities as muchless cells were stained red.

Fig. 4 also presents the results of tested cells under TEM. It can beobserved that normal E. coli cells exhibited a complete, smooth elon-gated structure. After treatment with GA-LIP, the E. coli cells collapsedand swelled, and wrinkles appeared on the surface of the cell mem-brane. When treated with LF-GA-LIP, the surface of the cells appearedsunken, and the contents were dissolved; some cells were severelybroken, as broken cell membrane fragments could be found, the con-tents were condensed to the cell wall, and a large area of the electron-transparent area was apparent in the cell, and the cells were like emptyshells. Similarly, the control S. aureus were closely arranged in uniformsizes, with a typical grape globular shape. However, the morphology ofthe cells treated with GA-LIP and LF-GA-LIP had changed. The contentsoverflowed; no cell debris were observed, the cell wall was severelydamaged and the bacteria disintegrated. For comparison, gallic acidand lactoferrin treatment led to less morphological changes than theliposomes, suggesting the higher antibacterial activities of the lipo-somal gallic acid.

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4. Conclusion

This study provides a better understanding of the effect of lacto-ferrin surface decoration on the physiochemical properties of gallic acidloaded liposomes. The electrostatic interaction between lactoferrin andphospholipid contribute to the decoration. Gallic acid existed in anamorphous state in the liposomes. LF-GA-LIP exhibited a more fa-vourable storage stability and a higher antibacterial activity comparedwith GA-LIP. The delayed-release effect could be also achieved by thelactoferrin surface decoration on the GA-LIP. These properties increasedthe potential of lactoferrin surface decorated liposomes as an effectivedelivery system for nutraceuticals in functional foods.

Acknowledgment

The authors thank National Nature Science Foundation of China(No. 31501578), University Science and Technology Project ofShandong Province of China (No. J16LE22), Doctoral ScienceFoundation of Shandong Province of China (No. BS2015SW019),Special Funds for Taishan Scholars Project of Shandong Province ofChina (No. ts201712058), and Advanced Talents Foundation ofQingdao Agricultural University (No. 6631115030) for financial sup-port.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2019.04.116.

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