DPPG Langmuir monolayers

Accepted Manuscript The Effect of Chitin Nanoparticles on Surface Behavior of DPPC/DPPG Langmuir Monolayers Ruijin Wang, Yi Guo, Hengjiang Liu, Yuning Chen, Yazhuo Shang, Honglai Liu PII: DOI: Reference:

S0021-9797(18)30161-9 https://doi.org/10.1016/j.jcis.2018.02.021 YJCIS 23293

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

9 January 2018 4 February 2018 5 February 2018

Please cite this article as: R. Wang, Y. Guo, H. Liu, Y. Chen, Y. Shang, H. Liu, The Effect of Chitin Nanoparticles on Surface Behavior of DPPC/DPPG Langmuir Monolayers, Journal of Colloid and Interface Science (2018), doi: https://doi.org/10.1016/j.jcis.2018.02.021

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The Effect of Chitin Nanoparticles on Surface Behavior of DPPC/DPPG Langmuir Monolayers Ruijin Wanga, Yi Guoa, Hengjiang Liua, Yuning Chenb, Yazhuo Shanga,*, Honglai Liu a a

Key Laboratory for Advanced Materials, School of Chemistry & Molecular

Engineering, East China University of Science and Technology, Shanghai 200237, China b

Ulink College of Shanghai, Shanghai 200237, China

*Corresponding author: [email protected] (Y. Z. Shang). TEL & FAX: 86 21 6425 2767

Abstract The effect of chitin nanoparticles on surface behavior of lipid systems containing dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) is studied by surface pressure (π)-area (A) isotherms, polarization-modulation infrared reflection absorption spectroscopy (PM-IRRAS), Brewster angle microscopy (BAM). The variation of surface behavior of DPPC/DPPG monolayers is induced mainly by electrostatic interactions between nanoparticles and head groups of phospholipids. At lower surface pressure, nanoparticles can penetrate into the monolayers and the positive charges carried by nanoparticles benefits the enrichment of phospholipid molecules at surface, which not only increases the mean molecular area but also hinders the formation of phospholipid liquid-condensed (LC) phase. However, when surface pressure is higher, the nanoparticles flee away from the surface and some of the phospholipid molecules are pulled out of the monolayers together to the subphase and decrease the order degree of the monolayers. Moreover, nanoparticles can destroy the hydrogen-bond between water molecules and phosphate head groups and thus lead to the dehydration of phosphate groups. This work confirms that chitin nanoparticles can affect the surface behavior of DPPC/DPPG monolayers. Furthermore, the results obtained using mixed monolayer containing two major lung surfactants DPPC/DPPG and nanoparticles will be helpful for deep understanding the harm of PM2.5 to lung health.

Keywords: Chitin nanoparticles; Lung surfactants; DPPC/DPPG monolayers; Surface behavior

1. Introduction In recent years, the air pollution is becoming more and more serious with the development of industry [1-3]. Fine particulate matter (PM2.5) is a significant air pollutant that consists of inorganic ions, metals, polycyclic aromatic hydrocarbons and other particles of 2.5 µm or less in diameter [4]. Owing to its small size, PM2.5 has a longer suspension time and a further propagation distance in the atmosphere, so people has a more widespread exposure level to PM2.5 [5]. As we all known that PM2.5 can enter the lung and even reach the alveolar region, causing respiratory, cardiovascular and cerebrovascular diseases [6-8]. By far, numerous studies have shown that PM2.5 still poses a nontrivial risk to public health even at far below national standard levels [9]. Lung is the respiratory organ of the human, and alveolar in the lung are responsible for gas exchange. The alveolar surface covered with a layer of lipid secretion, called Lung Surfactant (LS). LS is a complex mixture of phospholipids (80%), neutral lipids (8-10%), and surfactant associated proteins (10%) to regulate the alveolar surface tension [10-12] and a model involved dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) is widely adopted. LS are not only the important material guarantee of human normal respiratory function, but also play an important role in the immune system of the lung. LS monolayers are the first barrier that nanoparticles enter body, understanding the interaction mechanism of nanoparticles and LS monolayers is very important. However, it is difficult to estimate the effect of nanoparticles on LS monolayers directly in vivo, Langmuir monolayers on the air-water surface can be a good model to mimic the LS behavior and its interaction with nanoparticles. The Langmuir film technique on the air-water surface is a typical two-dimensional (2D) surface chemistry approach, widely applied for the structure and property studies of amphiphilic molecular at the interface, such as surfactants, polymer, proteins, and lipids [13]. The conformation and molecular interactions at the surface can be controlled and tuned by adjusting area or by altering the components of the subphase solution. Guzmán and coworkers gave a systematical

and profound study on the interaction between lipid monolayers and nanoparticles, hydrophilic silica [14-20] and titanium dioxide [21], they found that nanoparticles incorporate into the monolayers driven by the electrostatic interactions or hydrogen bonds depending on the nature of the lipids involved [22]. Besides, the hydrophobic silica [23, 24] and carbon black particles [25] also affect the monolayers interface behaviors depending on the relative quantity of lipids and nanoparticles spread on the air-water interface. Dwivedi et al. found that nanoparticles with different diameters, 12 nm and 136 nm, have contrasting effect on the functional and structural behavior of LS monolayers [26]. Melbourne et al. studied the interaction between LS monolayers and multiwall carbon nanotubes (MWCNTs) and a length-dependent can be found. The ‘shorter’ MWCNTs (1.1, 2.1µm) can penetrate the monolayers and ‘longer’ MWCNTs (35µm) are squeezed out of the monolayers during compression [27]. Sun et al. studied the interaction between Fe3O4 nanoparticles and DPPC monolayers, proving that the interaction depended on concentration of Fe3O4 nanoparticles and surface pressure of monolayers [28]. However, the mechanism of nanoparticles induce respiratory system disease is controversial and further study to reveal the effect of nanoparticles on lung health is still necessary. Chitin, poly(β-(1-4)-N-acetyl-D-glucosamine), is the second most abundant biopolymer next to cellulose in nature [29]. Particulate matter of chitin in the atmosphere is mainly come from two processes, natural and anthropogenic. The former is from the extensive distribution of fungi, insects, helminths and arthropods, their shells, mainly composed of chitin, decomposed into fragments in the air by chitinase; the latter is from the industrial sources. Chitin has been gaining significant momentum over the past few years for its application in food, maquillage, and medical treatment due to its unique properties including low toxicity, biocompatible, and biodegradable properties etc [30-33]. Consequently, the widely application of chitin also provides people more opportunities to exposure to chitin. Unfortunately, recent study have shown that chitin can induce expression of three epithelial cytokines, interleukin-25 (IL-25), IL-33, and thymic stromal lymphopoietin (TSLP), which initiates innate type 2 lung inflammation [34]. The study on the effect of chitin on

lung of mice also shown that the chitin can lead to pulmonary fibrosis spontaneously [35]. Furthermore, a number of studies also found that chitin can cause multiple immune reactions in the human respiratory system [36-39]. Obviously, on the one hand chitin brings convenience to our life, but on the other hand it also affects our health, especially the health of the respiratory system. In the present work, DPPC/DPPG monolayers

on the air-water surface is

adopted to mimic the lung surfactant monolayers and the chitin nanoparticles are selected as the target to study the effect of chitin nanoparticles on the surface behavior of DPPC/DPPG monolayers by a variety of techniques, including surface pressure-area and compressibility modulus-surface pressure isotherms, relaxation isotherm, PM-IRRAS measurements as well as BAM observations. The effect mechanism of chitin nanoparticles on surface behavior of DPPC/DPPG monolayers is proposed. The results may provide useful information not only for deep understanding of the effect of chitin nanoparticles on surface behavior of DPPC/DPPG monolayers but also for further comprehension of the harm of PM2.5 to lung health.

2. Materials and Methods 2.1 Materials The chemical structure of lipids and chitin nanoparticles used in this work are shown in Fig. 1 Dipalmitoylphosphatidylcholine (DPPC, purity >98%), and dipalmitoylphosphatidylglycerol (DPPG, purity >98%) are purchased from Lipoid (Germany). Chitin nanoparticles are purchased as colloid from Guanghan Hengyu New Materials Co. Ltd. The nanoparticles has been characterized by dynamic light scattering (DLS) and ζ-potential measurements（D=50 nm, ζ=15 mV）. Chloroform (CHCl3, purity >99%) and methanol (CH4O, purity >99%) are purchased from Shanghai Titan Polytron Technologies Inc. Ultrapure water with a resistivity of 18.2 MΩ·cm from Millipore Simplicity water purification system, adjusting pH=7.4, is used for all experiments.

Fig. 1. The chemical structure of DPPC (A), DPPG (B) and Chitin (C). 2.2 Monolayers preparation A Langmuir minitrough (KSV NIMA, Finland) with an area of 243cm2 is equipped for the study. The surface pressure is monitored by a force transducer connected to an alloy wire probe with a sensitivity of ±0.01mN/m. DPPC, DPPG or DPPC/DPPG (4:1, mol/mol) are dissolved in chloroform/methanol (9:1, v/v) mixture and the concentration is about 0.5-1.0 mg/mL. Aliquot of this solution are spread carefully dropwise on the air-water surface using a 100 µL syringe (Hamilton). Then, a waiting time of 30 min is needed for solvent evaporation and Langmuir monolayers to reach equilibrium. Then the monolayers can be used for other measurement. All experiments are conducted in triplicate under 20 ± 0.5 ℃. 2.3 Surface Pressure (π)-area (A) isotherms To study the effect of the concentration of chitin nanoparticles on lipid monolayers, DPPC/DPPG is deposited dropwise on the surface of chitin aqueous dispersion with different chitin concentrations (0, 0.5, 1.0, 5.0, 10.0 mg/L). After 30 min, the monolayers are compressed with a constant rate of 10 mm/min, and each measurement was repeated at least three times. 2.4 Relaxation of DPPC/DPPG monolayers at constant molecular area The monolayers on pure water or 1mg/L chitin aqueous solution are compressed to the target surface pressure (5 and 30mN/m) with a rate of 10 mm/min, then the

monolayers relaxation progress of surface pressure vs time (π-t) is recorded at a constant molecular area, and repeat each measurement at least three times. 2.5

Polarization-modulation

infrared

reflection

absorption

spectroscopy

(PM-IRRAS) measurements PM-IRRAS measurement is conducted with a KSV PMI550 instrument (KSV NIMA, Finland). The incoming light beam reached DPPC/DPPG monolayers at a fixed angle of 76°, where has the best signal to noise ratio [40]. The polarization modulation method eliminates background signals coming from environmental factors such as water vapor and CO2. The PM units modulate the incoming light continuously between s and p polarization, and a highly sensitive MCT-detector is used to measure both polarizations. The difference between the spectra provided surface-specific information, and the sum provided the reference spectrum [41]. At least 1200 scans are necessary for each spectrum that is presented in the present study. The frequency of wavelength modulation is 1500 cm-1, and resolution is 8 cm-1. The surface pressure is maintained at 30mN/m by moving the barriers. All experiments are conducted in triplicate. 2.6 Brewster angle microscopy (BAM) A micro BAM (KSV NIMA, Finland) is used to study the morphology of the DPPC, DPPG and DPPC/DPPG monolayers. It is equipped with a 20 mW laser emitting p-polarized light of 659 nm wavelengths and the resolution is 8 µm-1. The technique is sensitive to the density and thickness of the film on the air-water surface. At the Brewster angle (53.15°), no reflected signal is detected on the bare air-water surface, but when a monolayers is formed on the surface, the reflect index changes, resulting the light reflected and detected by camera [42]. BAM images are obtained with the monolayers compression simultaneously.

3. Results and Discussion 3.1 Surface pressure (π)-area (A) and compressibility modulus ( C s−1 )-surface pressure (π) isotherms.

Fig. 2. Surface pressure (π)-area (A) isotherms of DPPC/DPPG (4:1, mol/mol ) monolayers spread on subphase of chitin aqueous dispersion with different concentrations. Molecules in monolayers can exist in different states as a function of density. They are generally classified as gaseous (G), liquid-expanded (LE), liquid-condensed (LC), solid (S), and intermediate or transition films, in many respects analogous to those in three-dimensional systems [43]. The molecular density increases with the increase of surface pressure, so a continuous π–A isotherm can be obtained during compression. The typical π-A isotherms of DPPC/DPPG monolayers spread on subphase of chitin aqueous dispersion with different concentrations are shown in Fig. 2. As can be seen that the π-A isotherms of the Langmuir monolayers don’t show obvious plateaus that corresponds to the two-phase coexistence region [44, 45]. At high pressure (>48 mN/m) the isotherms almost merge thus clearly showing that the chitin does not influence the structure of DPPC/DPPG monolayers at higher surface pressures no matter its concentration. With the decrease of surface pressure, a little expansion of the DPPC/DPPG monolayers is observed and the corresponding mean molecular area increases slightly. The effect of the chitin on the π-A isotherms are almost negligible. However, the effect of chitin becomes pronounced and the expansion of the monolayers becomes more obvious when the surface pressure is less than 25mN/m. The higher the chitin content, the more obvious of the monolayers expansion is. For the π-A isotherms of monolayers, there are three characteristic parameters,

the liftoff area AL (the molecular occupation area where the isotherm rising just emerges related to the baseline), the limiting area A∞ (an empirical parameter approximating the mean molecular cross sectional area), and the collapse pressure πC [43]. If a LC or solid phase is observed in the isotherm, the A∞ value is conventionally given by the extrapolation of the steepest portion of the π–A curves to π=0. A∞, AL, πC, and molecule area at collapse (AC) at different concentrations are listed in Table 1. Table 1 shows that when the monolayers is on the pure water surface, the AL is 72.8 Å2, which is consistent with the results of the literature [46]. As the chitin concentration increases, the isotherms AL increase significantly, indicating that the chitin nanoparticles can penetrate into the monolayers under the lower surface pressure. However, πC, and Ac do not exhibit an obviously difference with the increase of chitin concentration. These evidences suggest that the chitin nanoparticles should be squeezed out of the monolayers and back to the subphase when the surface pressure is higher. Correspondingly, the limiting area A∞ shows no obvious variation. The similar type of isotherm behavior which results from the nanoparticles penetrating and squeezing out of the DPPC monolayers are observed by Guzmán et al. [15, 20]. Table 1. A∞, AL, πC, and AC of DPPC/DPPG (4:1, mol/mol) monolayers spread on subphase of chitin aqueous dispersion with different concentrations. Cchitin/mg/L

AL/Å2

A∞/Å2

πC/mN/m

AC/ Å2

0

72.8

46.5

48.5

47.4

0.5

77.2

47.8

48.8

48.7

1.0

82.6

47.7

49.5

48.6

5.0

93.2

47.9

49.6

47.8

10.0

102.0

48.4

49.4

48.3

It is worth noticing that there is no obvious phase transition during compression as shown in Fig. 2, so it is impossible to obtain the surface phase state of the monolayers directly. However, the compressibility modulus C s−1 provides us an alternative way to determine the phase state of monolayers. The compressibility

modulus C s−1 can be calculated by the following equation [47]: -1

Cs = (−1 / A)(dA / dπ)

(1)

Generally speaking, the gaseous (G) films is formed when the value of C s−1 falls between 0-12.5 mN/m; C s−1 =12.5-50 mN/m and C s−1 =100-250 mN/m corresponds to liquid-expanded (LE) films and liquid-condensed (LC) films, respectively; C s−1 >250 mN/m is solid (S) films [47]. Fig. 3 provides the variation of

compressibility modulus with surface pressure of DPPC/DPPG (4:1, mol/mol) monolayers spread on subphase of chitin aqueous dispersion with different concentrations. It can be seen that the phase state of monolayers change from LE films to LC films with the increase of surface pressure. With the increase of chitin concentration in the system, the surface pressure needed for the transition from LE films to LC films increase obviously, indicating that chitin nanoparticles hinder the formation of LC phase.

Fig. 3. Compressibility modulus ( C s−1 )-surface pressure (π) isotherms of

DPPC/DPPG (4:1, mol/mol) monolayers spread on subphase of chitin aqueous dispersion with different concentrations. 3.2 The relaxation properties of DPPC/DPPG monolayers

For further understanding the effect of chitin on the stability of monolayers, a relaxation study is performed at surface pressure of 5 and 30 mN/m, where correspond to the LE phase and LC phase. Generally, there are two methods of

recording the relaxation process of monolayers, π-t and A-t relaxation. For the π-t measurement, the surface pressure recorded as a function of time at constant area (stopped barriers). For the A-t measurement, the surface pressure is maintained at the initial value by the continuous adjustment of the barriers position while the area A is recorded as a function of time [48]. In the present study, the stability of monolayers is evaluated by π-t relaxation. Fig. 4 provides the π-t relaxation curves (normalized to π/π0). The curves can be fitted well by using the following equation [49]:

π/π 0 = C + ae − t / τ

(2)

Where C could be defined as the normalized equilibrium pressure and τ could be regarded as the lifetime which is related to the reorganization of monolayers [49]. The values of C and τ are listed in Table 2. Table 2. C, a, τ, r2, obtained by fitting the decay curves to a single-exponential

equation. π/mN/m 30mN/m

5mN/m

Cchitin/mg/L

C

a

τ

r2

0

0.924

0.053

1.709

0.9624

1

0.907

0.067

2.178

0.9605

0

0.837

0.150

2.715

0.9865

1

0.870

0.121

4.184

0.9896

From the fitting results, we can see that the lower of the surface pressure, the longer of the monolayers relaxation time, this is because the disorder degree of monolayers is higher at a lower surface pressure, so the time of conformation transition is longer. Interestingly, when the mean molecule area keep constant (53.19, 58.04 Å2/molecular for monolayers spread on pure water and 1 mg/L chitin aqueous respectively, corresponding to the lower initial surface pressure of π0=5 mN/m), the equilibrium surface pressure of monolayers spread on subphase of chitin aqueous (1 mg/L) is larger than that on the pure water, this should be attributed to the penetration of chitin nanoparticles into the monolayers and the positive charges carried by chitin nanoparticles benefits the enrichment of phospholipid surfactants at surface. However, when the initial surface pressure is higher (41.84, 42.89 Å2/molecular for monolayers

spread on pure water and 1 mg/L chitin aqueous respectively, corresponding to the higher initial surface pressure of π0=30 mN/m), the chitin nanoparticles flee away from the surface and some of phospholipid molecules are pulled out of the monolayers together with chitin nanoparticles due to their electrostatic attraction and leading to the equilibrium surface pressure decrease. The surface properties of particles can be modified by the adsorption of lipid which influences their further fate in the respiratory system and in the organism (retention time, bioavailability, toxicity, etc.) [50].

Fig. 4. π/π0-t relaxation isotherms of DPPC/DPPG (4:1, mol/mol) monolayers at

constant surface area. 3.3 PM-IRRAS measurements

Fig. 5. PM-IRRAS spectra for DPPC/DPPG (4:1, mol/mol) monolayers spread on

pure water and 1 mg/L chitin aqueous when surface pressure keeps 30 mN/m. A: C-H stretching regions; B: phosphate stretching regions. Fig. 5 provides the PM-IRRAS spectra for the monolayers of DPPC/DPPG. Fig. 5A presents the region of C-H stretches in -CH2 and -CH3. The bands near 2843 cm-1 and 2915 cm-1 are ascribed to the symmetric and asymmetric stretching of -CH2, and the bands near 2881 cm-1 and 2963 cm-1 corresponding to the symmetric and asymmetric stretching of -CH3. With the addition of chitin, the stretching bands shift to higher wavenumber. At the same time, the ratio of the absorption peak intensity of -CH2 asymmetric to symmetric bands is decreased from 1.55 to 1.08, suggesting that the introduction of chitin nanoparticles in subphase disorders the close-packed structure of the monolayers [51]. The electrostatic interaction between chitin nanoparticles and lipids molecular modify the rearrangement of the chain at the interface, affecting the order degree of alkyl tail chain. The charges carried by nanoparticles may be the important disrupting agent of the ordering of monolayers [15]. Fig. 5B is the PM-IRRAS spectra of main bands for phosphate region of lipids. The bands near 1082 cm-1 and 1227 cm-1 are ascribed to the PO2- symmetric and asymmetric stretching, respectively. With the introduction of chitin nanoparticles to the subphase, the symmetric stretching band of PO2- shifts to 1086 cm-1, which may be the results of the dehydration of the phosphate [52]. It is obviously, the chitin nanoparticles are disadvantage to the formation of hydrogen bond between water

molecules and phosphate head groups of phospholipid surfactants due to the existence of a large number of alcoholic hydroxyl groups on the surface of chitin nanoparticles, which should be the main reason for the dehydration of phosphate groups. 3.4 In situ BAM observations

Fig. 6. BAM images of DPPC/DPPG monolayers (4:1, mol/mol) spread on subphase of chitin aqueous dispersion under different surface pressures (The chitin concentration in subphase: 0, 1, 5, 10 mg/L). Further characterization of the effect of chitin nanoparticles on the morphology of the mixed DPPC/DPPG monolayers is performed by using BAM. Fig. 6 shows the BAM images for the monolayers at different surface pressures with and without chitin

in subphase. All the monolayers display the same phase transition process from LE, undergoes LE-LC to LC phase. In the presence of chitin nanoparticles, however, the LC domain appears when the surface pressure is higher. At a surface pressure of 3mN/m, the area of LC domain decrease with the increase of chitin concentration in subphase significantly as observed in Fig. 6e-h. When the surface pressure is increased to 9 mN/m, the density of the LC domain decreases with the increase of chitin concentration (Fig. 6i-l). These results reconfirm that chitin hinders the formation of phospholipid LC domains by penetrating into the monolayers. Interestingly, the similarly phenomenon also be observed in the monolayers containing DPPC spread on hydrophilic and hydrophobic silica [14, 23], as well as titanium dioxide dispersion [21]. Although the studied nanoparticles have different surface characteristics and chemical component, they all are charged. These observations strongly support the hypothesis that the incorporation of charged particles in the monolayer alters the electrostatic interaction between lipids, as a consequence, the morphology of the layer [21]. It is worth noticing that the BAM pictures depend on the rate of compression of the monolayers in some degree and thus it is difficult to interpret it accurately. However, the general consensus for lipid monolayers domains is that the shape of domain is determined by the competition between line tension and electrostatic interactions, the former favoring rounded domains and the latter favoring more elongated domains [53]. Unfortunately, the changes in shape of domains cannot be observed completely due to the poor resolution of the instrument (8µm-1). With the addition of chitin nanoparticles to the subphase, the visible LC domain become smaller and smaller until disappear. Obviously, the introduced positively charged nanoparticles on one hand enhance the density of phospholipid surfactants at the air-water surface and resulting in a decrease in line tension, on the other hand decrease the electrostatic repulsion force among negatively charged phospholipids. Maybe the decrease of linear tension is overwhelming, suggesting the functional dominance of electrostatic interactions, rather than line tension, for the studied system. As a result, the domain shape translates from rounded to elongated domains. However,

the elongated domains are narrow enough and cannot be detected by the present BAM.

Fig. 7. BAM images of monolayer of DPPC or DPPG spread on subphase of pure water or chitin aqueous dispersion under different surface pressures. In order to further understand the effect of nanoparticles on mixed DPPC/DPPG monolayers, we investigated the effects of nanoparticles on the morphology of DPPC and DPPG monolayers respectively (Fig. 7). For DPPC monolayers, there is no obvious difference in the morphology under different surface pressures. However, a distinct difference is observed with the addition of chitin to the subphase for DPPG monolayers. On the pure water surface, DPPG monolayers from uniform “network-like” domain to a homogeneous LC phase with the increase of surface

pressure. When introducing the chitin in subphase, however, DPPG molecules appears irregular reunion, and the homogeneous LC phase is formed at a higher surface pressure about 6.2 mN/m. Obviously, the stronger electronegativity of DPPG than that of DPPC results in a stronger electrostatic interaction with positively charged nanoparticles. Based on the above analysis, the possible interaction mechanism between nanoparticles and phospholipid monolayers is proposed (Fig. 8). When the monolayers are spread on the pure water, the arrangements of phospholipid molecules change from disorder to order gradually with the increase of the surface pressure. However, when the chitin nanoparticles are present in subphase, the nanoparticles tend to locate at or around the air-water surface and the phospholipid molecular density improved due to the electrostatic attractive interaction between nanoparticles and phospholipid molecules. With the increase of surface pressure, the nanoparticles are squeezed out of the surface, which affects the order degree of tail chain. At the same time, due to the diffusion of nanoparticles in water, the phospholipid molecules which bound to the surface of nanoparticles are pulled into the water, resulting in the reduction of surface pressure.

Fig. 8. The possible surface behavior of DPPC/DPPG monolayers with and without chitin nanoparticles in subphase.

4. Conclusion In this study, the influence of chitin nanoparticles on surface behavior of lipid systems containing DPPC and DPPG (DPPC/DPPG=4:1, mol/mol) is systematically studied. The results of this work indicate that the surface behavior of DPPC/DPPG

monolayers is affected mainly by the electrostatic interactions between nanoparticles and head groups of phospholipids. When surface pressure is lower, the electrostatic attraction induces positively charged chitin nanoparticles penetrate into the negatively charged DPPC/DPPG monolayers and thus benefits the enrichment of phospholipid surfactants at surface. Correspondingly the mean molecular area and the phase transition pressure increase significantly and the formation of phospholipid LC phase is hindered. The chitin nanoparticles flee away from the surface together with some of the attached phospholipid molecules to the subphase when surface pressure is higher and therefore leading to the decrease of the order degree of the initial well-arranged monolayers. Furthermore, the introduction of chitin nanoparticles can also cause the dehydration of phosphate groups. The present work not only provides the possible effect mechanism of chitin nanoparticles on the surface behavior of DPPC/DPPG monolayers but also provide more detailed information for further understand the physiological toxicity of chitin nanoparticles to human lungs.

Acknowledgements This work is supported by the National Natural Science Foundation of China (Project No. 21476072) and the Fundamental Research Funds for the Central Universities.

Reference [1] E. Feizabad, A. Hosseinnezhad, Z. Maghbooli, M. Ramezani, R. Hashemian, S. Moattari, Archives of Osteoporosis 12 (2017) 34. [2] G.W.K. Wong, T.F. Leung, T.F. Fok, Food & Chemical Toxicology An International Journal Published for the British Industrial Biological Research Association 37 (2004) 318. [3] J. Lelieveld, J.S. Evans, M. Fnais, D. Giannadaki, A. Pozzer, Nature 525 (2015) 367. [4] J. Yan, C.H. Lai, S.C. Lung, C. Chen, W.C. Wang, P.I. Huang, C.H. Lin, Science of the Total Environment 599-600 (2017) 1658.

[5] X. Han, Y. Liu, H. Gao, J. Ma, X. Mao, Y. Wang, X. Ma, Science of the Total Environment s 607–608 (2017) 1009. [6] F. Dominici, R.D. Peng, M.L. Bell, M.L. Pham, A. Mcdermott, S.L. Zeger, J.M. Samet, Jama the Journal of the American Medical Association 295 (2006) 1127. [7] J. Schwartz, L.M. Neas, Epidemiology 11 (2000) 6. [8] H. Lin, L. Tao, J. Xiao, W. Zeng, L. Guo, L. Xing, Y. Xu, Y. Zhang, J.J. Chang, M.G. Vaughn, Journal of Exposure Science & Environmental Epidemiology 27 (2017) 333. [9] S. Feng, D. Gao, F. Liao, F. Zhou, X. Wang, Ecotoxicology & Environmental Safety 128 (2016) 67. [10] G. Hu, J. Bao, X. Shi, R.P. Valle, Q. Fan, Y.Y. Zuo, Acs Nano 7 (2013) 10525. [11] R. Wüstneck, J. Perez-Gil, N. Wüstneck, A. Cruz, V.B. Fainerman, U. Pison, Advances in Colloid & Interface Science 117 (2005) 33. [12] J. Goerke, Biochim Biophys Acta 1408 (1998) 79. [13] S. Li, J. Guo, R.A. Patel, A.L. Dadlani, R.M. Leblanc, Langmuir the Acs Journal of Surfaces & Colloids 29 (2013) 5742. [14] E. Guzmán, L. Liggieri, E. Santini, M. Ferrari, F. Ravera, Colloids & Surfaces A Physicochemical & Engineering Aspects 413 (2012) 174. [15] E. Guzmán, L. Liggieri, E. Santini, M. Ferrari, F. Ravera, Soft Matter 8 (2012) 3938. [16] E. Guzmán, L. Liggieri, E. Santini, M. Ferrari, F. Ravera, Colloids & Surfaces B Biointerfaces 105 (2013) 284. [17] E. Guzmán, M. Ferrari, E. Santini, L. Liggieri, F. Ravera, Colloids & Surfaces B Biointerfaces 136 (2015) 971. [18] E. Guzmán, D. Orsi, L. Cristofolini, L. Liggieri, F. Ravera, Langmuir the Acs Journal of Surfaces & Colloids 30 (2014) 11504. [19] D. Orsi, Sci Rep-Uk 5 (2015) 17930. [20] E. Guzmán, L. Liggieri, E. Santini, M. Ferrari, F. Ravera, Colloids & Surfaces A Physicochemical & Engineering Aspects 413 (2012) 280.

[21] E. Guzmán, E. Santini, M. Ferrari, L. Liggieri, F. Ravera, Langmuir the Acs Journal of Surfaces & Colloids 33 (2017) 10715. [22] E. Guzmán, L. Liggieri, E. Santini, M. Ferrari, F. Ravera, Journal of Physical Chemistry C 115 (2011) 21715. [23] E. Guzmán, E. Santini, M. Ferrari, L. Liggieri, F. Ravera, Journal of Physical Chemistry C 119 (2015) 21024. [24] D. Orsi, T. Rimoldi, n.E. Guzmán, L. Liggieri, F. Ravera, B. Ruta, L. Cristofolini, Langmuir 32 (2016) 4868. [25] E. Guzmán, E. Santini, D. Zabiegaj, M. Ferrari, L. Liggieri, F. Ravera, Journal of Physical Chemistry C 119 (2015) 26937. [26] M.V. Dwivedi, R.K. Harishchandra, O. Koshkina, M. Maskos, H.J. Galla, Biophysical journal 106 (2014) 289. [27] J. Melbourne, A. Clancy, J. Seiffert, J. Skepper, T.D. Tetley, M.S. Shaffer, A. Porter, Biomaterials 55 (2015) 24. [28] C. Hao, J. Li, W. Mu, L. Zhu, J. Yang, H. Liu, B. Li, S. Chen, R. Sun, Applied Surface Science 362 (2016) 121. [29] F. Shahidi, J.K.V. Arachchi, Y.J. Jeon, Trends in Food Science & Technology 10 (1999) 37. [30] J. Dutta, S. Tripathi, P.K. Dutta, Food science and technology international = Ciencia y tecnología de los alimentos internacional 18 (2012) 3. [31] I. Ito, T. Osaki, S. Ifuku, H. Saimoto, Y. Takamori, S. Kurozumi, T. Imagawa, K. Azuma, T. Tsuka, Y. Okamoto, Carbohydrate Polymers 101 (2014) 464. [32] A. Usman, K.M. Zia, M. Zuber, S. Tabasum, S. Rehman, F. Zia, International Journal of Biological Macromolecules 86 (2016) 630. [33] M. Rinaudo, Cheminform 38 (2007) 603. [34] S.J. Van Dyken, A. Mohapatra, J.C. Nussbaum, A.B. Molofsky, E.E. Thornton, S.F. Ziegler, A.N. Mckenzie, M.F. Krummel, H.E. Liang, R.M. Locksley, Immunity 40 (2014) 414. [35] S.J. Van Dyken, H.E. Liang, R.P. Naikawadi, P.G. Woodruff, P.J. Wolters, D.J. Erle, R.M. Locksley, Cell 169 (2017) 497.

[36] M. Kogiso, A. Nishiyama, T. Shinohara, M. Nakamura, E. Mizoguchi, Y. Misawa, E. Guinet, M. Nouri-Shirazi, C.K. Dorey, R.A. Henriksen, Journal of Leukocyte Biology 90 (2011) 167. [37] B. Koller, A.S. Müllerwiefel, R. Rupec, H.C. Korting, T. Ruzicka, Plos One 6 (2011) e16594. [38] S.C. Da, P. Pochard, C.G. Lee, J.A. Elias, American Journal of Respiratory & Critical Care Medicine 182 (2010) 1482. [39] R.M. Roy, H.C. Paes, S.G. Nanjappa, R. Sorkness, D. Gasper, A. Sterkel, M. Wüthrich, B.S. Klein, Mbio 4 (2013) 49. [40] D. Blaudez, T. Buffeteau, J.C. Cornut, B. Desbat, N. Escafre, M. Pezolet, J.M. Turlet, Applied Spectroscopy 47 (1993) 869. [41] T.E. Goto, L. Caseli, Langmuir the Acs Journal of Surfaces & Colloids 29 (2013) 9063. [42] W. Daear, P. Lai, M. Anikovskiy, E.J. Prenner, Journal of Physical Chemistry B 119 (2015) 5356. [43] C. Qibin, L. Xiaodong, W. Shaolei, X. Shouhong, L. Honglai, H. Ying, Journal of Colloid & Interface Science 314 (2007) 651. [44] C.S. Song, R.Q. Ye, B.Z. Mu, Colloids & Surfaces A Physicochemical & Engineering Aspects 302 (2007) 82. [45] H. Nakahara, S. Lee, G. Sugihara, C.H. Chang, O. Shibata, Langmuir the Acs Journal of Surfaces & Colloids 24 (2008) 3370. [46] R.K. Harishchandra, M. Saleem, H.J. Galla, Journal of the Royal Society Interface 7 Suppl 1 (2010) S15. [47] T. Hianik, C.L. Opstad, J. Sandorova, Z. Garaiova, V. Partali, H.R. Sliwka, Chemistry and physics of lipids 202 (2017) 13. [48] M.I. Viseu, A.M.G.D. Silva, S.M.B. Costa, Langmuir 17 (2001) 643. [49] X. Chen, J. Wang, N. Shen, Y. Luo, L. Li, M. Liu, R.K. Thomas, Langmuir 18 (2002) 6222. [50] T.R. Sosnowski, Current Opinion in Colloid & Interface Science 36 (2018) 1.

[51] J.V.N. Ferreira, T.M. Capello, L.J.A. Siqueira, J.H.G. Lago, L. Caseli, Langmuir the Acs Journal of Surfaces & Colloids 32 (2016) 3234. [52] E.M. Adams, C.B. Casper, H.C. Allen, Journal of Colloid & Interface Science 478 (2016) 353. [53] A. Aroti, E. Leontidis, E. Maltseva, G. Brezesinski, Journal Of Physical Chemistry B 108 (2004) 15238.

Figure captions: Fig. 1. The chemical structure of DPPC (A), DPPG (B) and Chitin (C). Fig. 2. Surface pressure (π)-area (A) isotherms of DPPC/DPPG (4:1, mol/mol )

monolayers spread on subphase of chitin aqueous dispersion with different concentrations. Fig. 3. Compressibility modulus ( C s−1 )-surface pressure (π) isotherms of

DPPC/DPPG (4:1, mol/mol) monolayers spread on subphase of chitin aqueous dispersion with different concentrations. Fig. 4. π/π0-t relaxation isotherms of DPPC/DPPG (4:1, mol/mol) monolayers at

constant surface area. Fig. 5. PM-IRRAS spectra for DPPC/DPPG (4:1, mol/mol) monolayers spread on

pure water and 1 mg/L chitin aqueous when surface pressure keeps 30 mN/m. A: C-H stretching regions; B: phosphate stretching regions. Fig. 6. BAM images of DPPC/DPPG monolayers (4:1, mol/mol) spread on subphase

of chitin aqueous dispersion under different surface pressures (The chitin concentration in subphase: 0, 1, 5, 10 mg/L). Fig. 7. BAM images of monolayer of DPPC or DPPG spread on subphase of pure

water or chitin aqueous dispersion under different surface pressures. Fig. 8. The possible surface behavior of DPPC/DPPG monolayers with and without

chitin nanoparticles in subphase.

Table captions: Table 1. A∞, AL, πC, and AC of DPPC/DPPG (4:1, mol/mol) monolayers spread on

subphase of chitin aqueous dispersion with different concentrations. Table 2. C, a, τ, r2, obtained by fitting the decay curves to a single-exponential

equation.

Graphical Abstract