Differential Interactions of Gelatin Nanoparticles with the Major Lipids

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Differential Interactions of Gelatine Nanoparticles with the Major Lipids of Model Lung Surfactant: Changes in the Lateral Membrane Organization Weiam Daear, Patrick Lai, Max Anikovskiy, and Elmar J. Prenner J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 06 Apr 2015 Downloaded from http://pubs.acs.org on April 7, 2015

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The Journal of Physical Chemistry

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Differential Interactions of Gelatine Nanoparticles

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with the Major Lipids of Model Lung Surfactant:

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Changes in the Lateral Membrane Organization

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a

Weiam Daear#, aPatrick Lai#, bMax Anikovskiy and aElmar J Prenner*

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a

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada

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b

Department of Chemistry, University of Calgary, Calgary, Alberta, Canada

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* Corresponding author: [email protected]

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#Authors contributed equally

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ACS Paragon Plus Environment

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ABSTRACT

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There has been an increasing interest in the potential of nanomedicine, particularly in the use of

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nanoparticles between 10 nm to 1 µm in diameter as drug delivery vehicles. For pulmonary drug

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delivery it is important to understand the effect of polymeric nanoparticles on the lung surfactant

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in order to optimize the carriers by reducing their potential toxicological effects. This work

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presents a biophysical study of the impact of gelatine nanoparticles on packing and lateral

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organization of simple and complex lipid layers containing the major components of lung

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surfactant. Zwitterionic phosphatidylcholines, negatively charged phosphatidylglycerols, and the

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sterol cholesterol were employed in the models. In addition, the impact of acyl chain length was

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investigated.

Packing was determined by surface pressure-area isotherms whereas direct

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imaging of the surfactant at the air-water interface was performed using Brewster angle

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microscopy. Our results indicate minor changes in the surface pressure-area isotherms but

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concomitantly significant effects on the lateral organization of the monolayers upon nanoparticle

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addition. The data also suggest differential interactions of nanoparticles with the major lipid

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classes. Gelatine nanoparticles interact stronger with negatively charged phosphatidyl-glycerols

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compared to zwitterionic phosphatidyl-cholines. Furthermore, charge distribution depending on

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the molar lipid ratio and acyl chain saturation is important as well. Even cholesterol, whose

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concentration is low compared to other components, plays an important role in nanoparticle

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interactions.

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KEYWORDS

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Pulmonary drug delivery, Lung surfactant models, Monolayer isotherms, Lateral domain

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architecture, Brewster angle microscopy


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1. INTRODUCTION

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The human body offers many routes that could be used for drug delivery. The pulmonary route,

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which is the direct delivery to/through the lungs, is of a particular growing interest. It is

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commonly used for the treatment of lung related diseases, but the advantages discussed below

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are also relevant for other treatment targets.

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Pharmaceutical compounds targeted to the lungs pass through the trachea and the large and small

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bronchioles before reaching the alveoli. The inner surface of the alveoli is a 'point of contact'

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barrier for any foreign substances including drugs. The goal in systemic drug delivery is to

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overcome this barrier to reach the blood circulation.

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The lungs provide a large surface area of 43-102 m1, 2 that increases drug adsorption in a low

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proteolytic environment2. In addition, the thin air-blood alveolar barrier of around 100 - 200 nm

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maximizes the relative transport of drugs3. Finally, the highly vascularized alveoli are very

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beneficial for local and systemic drug delivery2, 4, 5, 6.

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The alveolar epithelial layers mainly contain type I and type II cells covered by a thin aqueous

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layer of around 50 - 80 nm2 that is covered by lung surfactant (LS), which acts as the final

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barrier. LS is composed of 80% phospholipids, 5 - 10% neutral lipids, such as cholesterol, and 5

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- 10% proteins7, 8, 9. LS reduces the surface tension during expansion and compression of the

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monolayer during the breathing cycles and prevents lung collapse10, 11; moreover, it enables rapid

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film re-spreading upon inhalation10, 12. The major phospholipid component of the LS is

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dipalmitoyl-phosphatidyl-choline (DPPC) which constitutes about 30 - 45% of the

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phospholipids13, 14. This fully saturated rigid lipid is essential for film stability and for

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maintaining a low surface tension but does not contribute to film spreading15. This role has been


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attributed to unsaturated lipids such as the palmitoyloleoyl-phosphatidyl-glycerol (POPG) which

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increase film fluidity, facilitating film respreading upon exhalation16. In addition, LS contains 4

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surfactant proteins (SP) labeled A, B, C, and D. SP-A and SP-D are hydrophilic proteins that are

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responsible for regulating LS homeostasis as well as innate immune response12, 17, 18. On the

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other hand, SP-B and SP-C are hydrophobic, low molecular weight proteins that facilitate rapid

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LS lipid adsorption onto the air-water interface12, 19, 20.

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Recently, there has been growing interest for targeted and controlled pulmonary drug delivery

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with high efficacy and specificity21. This has led to the development of nanoparticles (NPs) with

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dimensions ranging from 1 nm to 800 nm21 to particles where at least one of the dimensions is

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below 100 nm22, 23. The advantages of using NPs as drug delivery vehicles lie in their ability to:

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1) permeate small blood capillaries, 2) facilitate controlled drug release, 3) provide protection

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against enzymatic degradation, and 4) stabilize volatile pharmaceutical compounds2, 21,24. NPs

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used in drug delivery can be made of a wide variety of materials such as lipids25, 26 or mixtures of

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different organic polymers27, 28.

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Use of NP based drugs also requires a better understanding of their potential impact on the lungs

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specifically with LS, which is the first barrier before drug transfer into the endothelial layer29.

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NP composition or surface properties could potentially impact the integrity of the LS. A previous

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study indicated moderate effects of the nanoparticle size on the extent of their interactions with

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LS, whereas all particle sizes between 136 - 287 nm induced a significant reduction of the

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surface potential30. This effect depends on the chemical nature and charge of the particles and

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has to be considered in drug delivery research. Biodegradable materials are of particular interest

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and, thus, the present work focuses on natural polymeric gelatine NPs and their interactions with


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the LS. Gelatine is obtained from natural sources such as collagen31 resulting in high

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biodegradability and low antigenicity, which are very attractive characteristics for drug delivery.

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A lot of work has been presented in the literature using biomimetic models for LS, in particular,

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DPPC30, 32, 33, 34, 35, 36, 37, 38, 39. DPPC represents the largest lipid component of the mammalian

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lungs (human, bovine, porcine) and is also a main component of clinical surfactant replacement

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systems37. Although this model is appropriate for humans, it may not apply to all mammals38.

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Monolayers at the air-water interfaces were used to assess the impact on film stability whereas

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Brewster angle microscopy (BAM) was used to image particle induced changes in the lateral

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film architecture. The work expands the previous investigation of DPPC-NP interactions39 by

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increasing the complexity of the lipid systems by using the biomimetic LS models based on the

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LS mass-spectrometry results40, 41.

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2. MATERIALS AND EXPERIMENTAL METHODS

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2.1 Materials

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All lipids were purchased from Avanti Polar Lipids Inc. (Alabaster, Alabama, USA). Gelatine,

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type B with a bloom of ~225, and 25% v/v glutaraldehyde was obtained from Sigma Aldrich

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(Oakville, Ontario, Canada). Chloroform, acetone, and methanol were purchased from EMD

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chemicals (Mississauga, Ontario, Canada) and were of ACS or HPLC grade.

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2.2 Methods

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2.2.1 Preparation and Characterization of Gelatine Nanoparticles


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Gelatine NPs were prepared using an optimized two-step desolvation method42. 1.25g of gelatine

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was dissolved in 25mL of deionized water at 40°C and 25mL of acetone was added dropwise

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while stirring at 600rpm. The latter step resulted in the precipitation of a high molecular weight

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fraction of gelatine. The supernatant was discarded and the precipitate was dissolved in 25mL of

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water. A drop-wise addition of 75mL of acetone resulted in the formation of the NPs. 250 µL of

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25% glutaraldehyde were added to crosslink the NPs and the suspension was continuously stirred

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for 16 hours at 600rpm. Acetone was removed using a rotary evaporator (Buechi R-200, Flawil,

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Switzerland) and the NPs suspension was stored at 4ᵒC. NPs were isolated using a

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microcentrifuge (ThermoFisher, Waltham, Massachusetts, United States) at 20,000xg for 45

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minutes. The nanoparticle pellets were redissolved in 6:4 ratio of chloroform:methanol and the

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concentration was determined by weighing the nanoparticles after drying a known volume of the

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nanoparticle suspension. In all experiments the ratio of NPs to lipids was 1 to 10 and all

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concentrations are express in mg/mL.

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2.2.2 Lipid Solutions

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All lipids were dissolved in a 6:4 v/v chloroform:methanol mixture to yield a concentration of

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1mM for the use on the Langmuir trough. The molar ratios of lipids used are shown in brackets

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(45) DPPC: (8) Palmitoyloleoyl-phosphatidylcholine, POPC: (1) Dipalmitoyl-

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phosphatidylglycerol, DPPG: (6) POPG. In addition, the neutral lipid cholesterol was added at a

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2% of the total lipid weight.

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2.2.3 Surface Pressure-Area Isotherm Stability Measurements

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Isotherms at the air-water interface were recorded using a pressure sensor (Type PS) equipped

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with a Wilhemy plate43 and a 660 x 10 cm teflon Langmuir trough (Biolin, Stockport, UK). 400


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mL of deionized water was used for the subphase. Compression was started after 10 minutes to

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allow solvent evaporation and was performed at a rate of 100 cm²/min by using two Teflon

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barriers. All experiments were performed at ambient conditions (25°C) and repeated at least 3

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times to ensure reproducibility. Error bars fall within the width of the line and are not included in

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the surface pressure-area isotherm figures.

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2.2.4 Brewster Angle Microscopy

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Brewster Angle Microscopy (BAM) allows direct visualization of the lateral organization of

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monolayers in real time. BAM is based on the fact that plane-polarized light directed at an air-

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water interface at the Brewster angle (~53.1 degrees for water) is not reflected. Once a

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monolayer is deposited at the surface, the refractive index changes resulting in the light being

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reflected and detected by a camera. Thus, the method allows real-time and label free

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visualization of monofilms44, 45. The Brewster Angle microscope and the required software EP3

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was provided by Accurion (Göttingen, Germany).

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2.2.5 3D Visualization

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Images for the pure DPPC and DPPC + NPs were obtained using EP3 view software described

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elsewhere46. Briefly, the lipid monolayer thickness can be estimated qualitatively from signal

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intensity at the z-axis. An increase in the intensity corresponds to an increase in the peak height

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and brightness.

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3. RESULTS AND DISCUSSION


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The use of NPs for the treatment of lung related diseases is pharmaceutically attractive and is

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being increasingly investigated47, 48, 49, 50. Nonetheless, it is unclear how the presence of NPs

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affects LS function during pulmonary drug delivery. As a result, a biophysical characterization

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of the impact of NPs on LS stability is relevant. Here we characterize the interactions between

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NPs and surfactant by investigating the role of different lipid species.

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3.1 Gelatine NP Characterization

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The gelatine NPs used have a hydrodynamic diameter of 107 nm with a polydispersity index of

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0.15 and a zeta potential value of +32 mV determined using a ZetaSizer Nano ZS (Malvern,

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Worcestershire. UK.) at pH 742.

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3.2 Surface Pressure-Area Isotherms

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3.2.1 Phosphatidylcholine Based Systems

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Figure 1 presents the pressure-area isotherms for the saturated and unsaturated

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phosphatidylcholines; Panel-A shows data for the fully saturated DPPC which is consistent with

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previously published results40, 51. At high molecular areas, the lipids are in the gas phase with

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limited interactions between lipids. As the monolayer is compressed to an area of ~ 95

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Å²/molecule, the isotherms lifts off the zero pressure line and the lipid packing changes to a so-

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called liquid-expanded (LE) phase which is characterized by increased interactions. However,

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the packing is still relatively fluid. A plateau is seen between 75 - 60 Å²/molecule at a surface


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pressure of 6 mN/m. This plateau is characteristic of DPPC isotherms corresponding to the co-

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existence of both liquid-expanded/liquid-condensed (LE/LC) phases. The isotherm collapses at

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an area of 37 Å²/molecule and at a surface pressure of 58 mN/m, at which point multilayers start

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forming52, 53.

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The addition of gelatine NPs to the system results in a slightly earlier take off at a molecular area

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of 105 Å²/molecule indicating that molecules in the film require a larger surface area. This

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finding suggests that gelatine NPs interact with the lipid head group. Upon further compression

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for the DPPC + NPs system, a small 'shoulder' is observed at a surface pressure between 8 and 16

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mN/m that is characteristic for phase co-existence. Upon even further compression, the area at

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the collapse pressure is reduced to about 45 Å²/molecule (a 5 Å²/molecule difference compared

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to pure DPPC) whereas the collapse pressure is reduced to 56 mN/m (2 mN/m difference

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compared to pure DPPC). The observed area reduction suggests that a fraction of lipids interact

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with the NPs, with limited impact on the film stability. The fact that film stability is maintained

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is an essential aspect for potential drug delivery applications. Similar results were observed

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previously39. It is important to note that the area/molecule values measured in the isotherm do

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not account for the molecular weight of the NPs. Thus, the observed shifts can only be treated as

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relative changes induced by NP addition. In addition, it appears impossible to determine the

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fraction of NPs within the monolayer, and, thus, the average molecular weight. A 10% w/w

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concentration of nanoparticles is higher compared to what would be observed during

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inhalation54.

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Figure 1 (Panel-B) shows the surface pressure-area isotherm for the POPC and POPC + NPs

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systems. The obtained POPC isotherm is in agreement with the literature55. The slow rising

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slope indicates LE phase formation throughout the entire compression range. This behavior is


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expected for mono-unsaturated lipids56 as the double bond limits packing and leads to the

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formation of more fluid films. The POPC monolayer collapses around 48mN/m, which is similar

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to the reported value55. The co-deposition of gelatine NPs does not change the shape, slope or

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collapse pressure of the isotherm.

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Figure 1 (Panel-C) shows the pressure-area isotherm for the binary PC mixtures containing

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DPPC and POPC at a molar ratio of 45:8 40 in the absence and presence of NPs. The addition of

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a 15% mol ratio of POPC to DPPC still has a strong impact on lipid packing as the characteristic

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DPPC plateau at 8 mN/m is significantly reduced in the mixture. Minor deviations to smaller

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areas just prior to collapse suggest a limited destabilization of the films by the NPs. The

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characteristic plateau seen for DPPC is only maintained as a shoulder in the curve at 8 mN/m and

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the subsequent increase in pressure around 12 mN/m is delayed in the presence of NP to 18

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mN/m. The isotherm slope of the mixture is similar to the one observed for pure DPPC

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suggesting tighter packing. Moreover, the LC phase formation is not significantly affected by the

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addition of NPs. The overall film stability assessed by collapse pressure is not affected by the

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addition of NPs to the PC containing systems, in agreement with previous data for DPPC39.

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Figure 1. Surface pressure-area isotherms of DPPC (A), POPC (B), binary PC (C) systems. The binary system has a molar ratio of 45:8 of DPPC:POPC. NPs co-spread in a film containing lipid and NPs at a ratio of 10:1 w/w in 6:4 Chloroform:Methanol solvent. Solid lines (──): lipid system, Dotted lines (···): lipid system + gelatine NPs. Figure insets indicate the location of shoulders in the isotherm for DPPC /+NPs and binary PC /+NPs systems. n =3

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3.2.2 Phosphatidylglycerol Based Systems

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Subsequently, the second most prevalent lipid class of LS, negatively charged PGs, has been

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investigated. The isotherm for the pure DPPG (Figure 2 Panel-A) is in agreement with

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previously published results57, 58 whereby the steep slope is consistent with the behavior of lipids

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containing fully saturated chains. The collapse pressure for the pure DPPG lipid system is 57

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mN/m. The addition of gelatine NPs has minor effects on the shape of the isotherm - there is a

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delay in the isotherm between the zero pressure line and 15 mN/m. This change caused by the

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NPs could be due to charge-charge interactions and insertion of the positively charged NPs

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between the negatively charged DPPG lipids. Furthermore, the NPs induce a reduction of the

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film stability indicated by a decrease of collapse pressure by 7 mN/m.

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The pressure-area isotherms for the partially unsaturated POPG with and without NPs are shown

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in Figure 2 (Panel-B). The POPG isotherm is similar to the previous results57. Both systems

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show a gentle slope increase characteristic for a LE phase as expected for lipids containing

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unsaturated acyl chains49. Both isotherms do not show a significant change up to a surface

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pressure of 12 mN/m. Above this surface pressure, the isotherm of the POPG + NPs shifts

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slightly to lower areas suggesting lipid-NP interaction. It is possible that NPs protruding the

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surface remove some of the lipid molecules from the monolayer, which, in turn, results in a

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decrease in surface pressure. Since the NPs are much bigger than the size of individual lipids,


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one particle may accommodate a large number of lipids. Both systems collapse at a surface

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pressure of 47 mN/m.

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The surface pressure-area isotherms for the binary PG systems containing DPPG and POPG with

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a molar ratio of 1:6 are shown in Figure 2 (Panel-C). This system is primarily unsaturated, and,

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thus, is expected to show trends similar to unsaturated systems. The pure binary PG system lifts

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off from the zero pressure line at an area of 110 Å²/molecule. An inflection is seen at a surface

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pressure of 15 mN/m corresponding to LE/LC phase co-existence. The binary PG + NPs system

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lifts off from the zero pressure line at a surface pressure of 115 Å²/molecule entering the LE

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phase. The NPs cause a slight deviation to smaller areas up to a surface pressure of 15 mN/m. An

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inflection is also seen for the binary PG + NPs system at a surface pressure of 23 mN/m

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corresponding to the stabilization of co-existing LE/LC phases. Both systems collapse at a

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pressure of 48 mN/m.

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Figure 2. Surface pressure-area isotherms of DPPG (A), POPG (B), binary PG (C) systems. The binary system had a molar ratio of 1:6 of DPPG:POPG. NPs co-spread in a film containing lipid and NPs at a ratio of 10:1 w/w in 6:4 Chloroform:Methanol solvent. Solid lines (──): lipid system, Dotted lines (···): lipid system + gelatin NPs. Figure inset indicates the location of shoulders in the isotherm for binary PG system. n=3

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3.2.3 Complex Systems

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In order to further improve the biomimetic model a quaternary mixture based on previous

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reports6,

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POPC, 1 DPPG and 6 POPG. Additionally, the quinary model containing a physiologically

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relevant 2% w/w of cholesterol61 has been studied. Such complex lipid models have been chosen

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to better mimic LS while still being able to characterize the role of individual lipids in LS-NP

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interactions. The isotherm of the quaternary systems is characterized by a more gentle overall

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slope due to the presence of ~ 25% monounsaturated PCs and PGs (Figure 3 Panel-A). The

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addition of NPs to the quaternary system results in almost overlapping isotherms, whereby minor

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inflections seen in the pure systems at a surface pressure of 10 mN/m are abolished. Above 25

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mN/m, the isotherms of the NPs containing systems shift to slightly lower areas. Such shifts are

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also observed for DPPC and POPG systems at high surface pressures and indicate a potential

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minor loss of lipids in the presence of particles either to the subphase bound to NPs or tighter

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packing induced by NPs. Another noteworthy feature is the gradual collapse observed in the

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quaternary system between surface pressures of 49 and 54 mN/m. This is also seen with the

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addition of NPs to the system from a surface pressure of 46 to 58 mN/m. The shape of this

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section of the isotherm corresponds to the “squeezing out” of the unsaturated fluid lipids. This

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also occurred in the same pressure range for the individual unsaturated lipids. The remaining

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saturated lipids are responsible for maintaining the low surface tension and the overall stability

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of the film. This phenomenon captured broad attention in the pertinent literature11, 62, 63, 64, 65. This

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theory suggests that during the compression of the lung surfactant the more rigid saturated lipids

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such as DPPC remain in plane and the more fluid unsaturated lipids form bends. The surfactant

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proteins SP-B and SP-C were found to be involved in the folding of the monolayer during

23, 40, 59, 60

has been studied. This system is made up of a molar ratio of 45 DPPC, 8


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compression which might have prevented the squeezing-out the unsaturated components57, 64, 65,

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66

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inhalation34.

. These unsaturated and fluid components are important for film spreading and adsorption upon

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For the quinary (DPPC, POPC, DPPG, POPG, and 2% w/w Cholesterol) and quinary + NPs

6

systems, the pressure-area isotherms are seen in Figure 3 (Panel-B). The addition of the natural

7

LS component cholesterol to the system does not change the isotherms of the quinary system

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(Figure 3 Panel-B). The inflection at a surface pressure of 10 mN/m is observed with and without

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the NPs indicative of remnants of LE-LC phase co-existence due to the high DPPC content. The

10

pure quinary system collapses between 49 and 54 mN/m. The addition of NPs slightly increases

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the range (46 to 60 mN/m).

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Figure 3. Surface pressure-area isotherms of quaternary systems constituting of molar ratio of 45 DPPC, 8 POPC, 1 DPPG, and 6 POPG (A) and quinary system constituting of 45 DPPC, 8 POPC, 1 DPPG, 6 POPG, and 2% of total weight cholesterol (B) systems. NPs co-spread in a film containing lipid and NPs at a ratio of 10:1 w/w in 6:4 Chloroform:Methanol solvent. Solid lines (──): lipid system, Dotted lines (···): lipid system + gelatin NPs. Figure insets indicate the


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location of shoulders in the isotherm for the quaternary and quinary systems. (*) indicate the start of system collapse. n=3

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3.3 Brewster Angle Microscope

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3.3.1. Phosphatidylcholine Based Systems

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In general, the addition of the NPs does not have a significant impact on the monolayer

7

isotherms which was consistent with previous reports on gelatine NP interactions with DPPC

8

model systems34, 39. Nevertheless, changes in the lateral organization of the monofilms were

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previously reported in the literature23, 30. Thus, Brewster angle microscopy (BAM) is used to

10

visualize the lateral organization of these model films in the presence and absence of NPs.

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Images of DPPC films are taken at 7 mN/m which is within the pressure range of the isotherm

12

plateau indicating phase co-existence, and at 30 mN/m, which is well below the collapse for all

13

systems, to allow for comparison between the different lipid mixtures (Figure 4 Panel-A). The

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observed LC domains with fractal-like shapes are consistent with previous results67, 51. These

15

domains initially start as circular domains that quickly increase in size and branch. DPPC

16

domains can exhibit many diverse shapes which are affected by different factors such as the

17

compression speed67, 68 as well as the incubation time for solvent evaporation. Generally, the

18

observed domain shapes are mainly controlled by two important competing forces; line tension

19

and electrostatic dipole-dipole repulsion69, 70. Line tension tends to favor the stabilization of

20

circular domains while electrostatic repulsion favors non-circular domains69. At a surface

21

pressure of 15 mN/m, the monolayer starts coalescing into a more homogenous film that was

22

maintained at higher surface pressures until collapse. When gelatine NPs are co-deposited

23

(Figure 4 Panel-B), smaller domains form at the isotherm kink region of 10 mN/m. Branching is


15


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Page 16 of 36

1

also seen in those domains but is less defined and elongated as for DPPC alone. The bright spots

2

or lipid-NP clusters are found to be either in the center or along the edges of the lipid domains.

3

With further compression, the domains start to coalesce into a homogenous film while the lipid-

4

NP clusters remain apparent until collapse pressure is reached. The large elliptical object in the

5

image (see arrow in panel A and B) is an artifact from the polarizer. The NPs containing sample

6

exhibits a significantly higher number of domains that are smaller in size at low pressures.

7

Moreover, NPs prevent the formation of a homogeneous film and the domains are observed until

8

a surface pressure of 15 mN/m.

9

The monounsaturated POPC does not result in LC domain formation across the entire pressure

10

range, due to limited packing in the fluid films (Figure 4 Panel-C). The lipid film appears to be

11

homogenous throughout the compression. Nonetheless, the BAM images of POPC in the

12

presence of NPs show bright clusters starting at 15 mN/m as seen in the figure insets of the left

13

image in Figure 4 (Panel D). With further compression, at a surface pressure of 30 mN/m, the

14

film is no longer homogenous and more lipid-NP clusters appear. Furthermore, compression of

15

the monolayer with NPs results in a significant lateral reorganization as three distinct features are

16

seen at 30 mN/m: 1) fluid LE phase (darker grey area), 2) more rigid LC phase (lighter grey),

17

and 3) NP induced protruding clusters seen as bright spots (Figure 4, panel-D, right). The

18

clusters of gelatine NPs appear to favor the rigid LC over the more fluid LE phase. More

19

experiments are needed in order to better understand these interactions.

20

The imaging of the binary mixture, with 15% mol ratio of monounsaturated POPC exhibited LC

21

domains comparable to DPPC shapes described above, although higher domain density and

22

larger domain sizes are observed. The significant impact of lipid composition was described in

23

detail for binary mixtures of rigid egg sphingomyelin and fluid POPC elsewhere71. The presence


16

Page 17 of 36

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1

of the larger POPC partly prevents optimal DPPC packing and, thus, the formation of a

2

homogeneous film at higher pressures (Figure 4 Panel E). When gelatine NPs are co-deposited, a

3

major decrease in the LC domains size is observed at comparable surface pressures (Figure 4

4

Panel-F). In addition, clusters within or at the edges of the domains are observed that are

5

attributed to the presence of NPs (Figure 4 Panel F). These clusters already form at low pressures

6

and do not disappear upon compression, although their size is diminished at 46 mN/m, which is

7

close to the collapse pressure. Nevertheless, no difference in collapse pressure in the presence of

8

NPs is observed for the isotherms (Figure 2 Panel C).

9

We assume that those bright spots in the BAM images correspond to lipid-NP clusters. These

10

clusters are not observed in the pure lipid systems and, thus, are due to the presence of NPs.

11

BAM generates images based on light being reflected from the film. Objects that protrude higher

12

out from the plane appear brighter. This allows generating a cross section of the monolayer and

13

determines the relative heights of the features present. Since the much brighter clusters are only

14

visible in the presence of NPs they are interpreted as lipid-NP clusters.


17


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Page 18 of 36

1 2 3 4 5 6

Figure 4. BAM images of PC systems; DPPC (A), DPPC + NPs (B), POPC (C), POPC + NPs (D), binary PC of molar ratio of 45 DPPC to 8 POPC (E), and binary PC + NPs (F). The black arrows in panels (A and B) is due to an artifact in the polarizer. White arrows in panel D and F represent lipid-NP clusters pointing to a region which magnified (2X) in the insert. The ratio of lipids to NPs is 10:1 w/w. Images are 271x219 microns. Bar is equal to 50 microns. n≥2

7 8

3.3.2 Phosphatidylglycerol Based Systems


18

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1

Subsequently, the lateral organization of PG containing monolayers has been investigated

2

(Figure 5). BAM images of DPPG (Figure 5, Panel-A) show that lipid domains form at low

3

surface pressures and quickly coalesce at ~ 10 mN/m to form a homogenous film which is

4

similar to previous reports on model systems72. At high surface pressures of ~30 mN/m and

5

before the collapse pressure is reached, brighter clusters are observed within the monolayer. This

6

could potentially indicate the formation of multilayers from the monolayer collapse as observed

7

by other groups51, 73. Similar domains are seen at ~10 mN/m upon the addition of gelatine NPs

8

(Figure 5 Panel-B). Nonetheless, a distinct difference is observed at higher surface pressures

9

where gelatine clusters appear to form at the edges of the PG lipid domains. Interestingly, in

10

contrast to the PC systems, these clusters are outside of the LC domains. This is by far the

11

strongest change in lateral film organization recorded (Figure 5, panel B), which suggests that

12

additional forces could be involved between NPs and the negatively charged PGs. Due to the

13

slightly positive charge of the NPs at these experimental conditions42 there will be electrostatic

14

attraction to the negative charge of PGs driving particles closer to the monolayer.

15

Similarly to the POPC system, POPG does not form any domains but forms a homogenous film

16

throughout the entire compression until collapse pressure is reached (Figure 5 Panel-C). This

17

behavior is similar to the one previously reported for POPG monolayers74. In contrast, the BAM

18

images for the NP containing POPG system show the formation of bright clusters at a pressure of

19

~15 mN/m in contrast to very few NP induced clusters in POPC. At higher pressures, many

20

small clusters are observed that are less bright than the ones seen in POPC. In some cases (see

21

arrow in panel D) darker structures appear that resemble more voids within the film which

22

indicate packing problems within the film and even early collapse; although these do not affect

23

the overall collapse pressure measured in the isotherm (see Figure 2 Panel B). These void-like


19


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Page 20 of 36

1

defect structures were reported in various forms such as lines or points for DPPC75 as well as

2

other systems such as DPPG and DPPS76.

3

These data suggest that the interactions are not only driven by charge-charge attractions but other

4

factors, such as charge distribution, differences in lipid packing (POPG vs. DPPG), or chemical

5

structure of the head group (POPC vs. POPG), are also relevant. For the binary PG mixture

6

which only contained ~ 15% DPPG, no domains are observed at lower pressures as seen before

7

for pure POPG (Figure 5 Panel E). Nevertheless, upon further compression, LC domains

8

presumably enriched in DPPG are formed. Compared to pure DPPG, they are smaller in size and

9

branched compared to the fairly round domains of the fully saturated lipid system. The binary

10

PG + NPs system (Figure 5 Panel F) show the presence of some brighter clusters similar to the

11

previous NP containing PG systems. At higher pressures, fewer clusters (see inset in panel F) but

12

darker potentially void-like structures occur suggesting a significant impact of NPs on the

13

packing of the binary film. No change in the collapse pressure was recorded in the monolayer

14

isotherms (see Figure 2 Panel C) but this does not exclude the possibility that the collapse

15

pressure will be affected after multiple compression-expansion cycles. The lipid-NP clusters

16

observed in the BAM images change the overall lateral domain organization which could result

17

in the film destabilization, i.e. a decrease in the collapse pressure, with time. Experiments with

18

multiple compression-expansion cycles can shed light on the role of the cluster formation on the

19

film stability.


20

Page 21 of 36

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1 2 3 4 5 6

Figure 5. BAM images of DPPG (A), DPPG + NPs (B), POPG (C), POPG + NPs (D), binary PG of molar ratio of 1 DPPG to 6 POPG (E), and binary PG + NPs (F). The arrow in panels (D) and (F) with figure insets indicate the appearance of film void. The inset in panel D is 2x magnification. The ratio of lipids to NPs is 10:1 w/w. n≥2

7

3.3.3 Complex Systems

8

Next, the more complex quaternary mixture is analyzed. Domains in this system appear at a

9

surface pressure of 10 mN/m and slightly increase in size upon compression (Figure 6 Panel-A).


21


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Page 22 of 36

1

This is similar to DPPC, which is the main component of these systems. These domains coalesce

2

at 25 mN/m into a homogenous LE film until the collapse pressure is reached similar to DPPC.

3

The more complex systems show better mixing compared to the binary PCs and PGs (see

4

Figures 4 and 5, panels E & F). This film mixing at higher surface pressures after domain

5

formation and growth was analyzed by other groups75. It was suggested that increased

6

homogeneity is due to the density similarity of the two lipid phases; LE and LC, and eventually

7

one becomes increasingly predominant over the other77.The addition of gelatine NPs results in

8

slightly more pronounced domain formation at similar surface pressures when compared to pure

9

quaternary system (Figure 6 Panel-B). Other components such as POPG may enhance domain

10

formation in the mixture.

11

The addition of cholesterol at 2% w/w to the quaternary lipid model induces significant changes

12

in the lateral lipid organization in terms of domain formation propensity and size (Figure 6 Panel

13

C). The quinary system at a surface pressure of ~ 12 mN/m exhibits smaller domains compared

14

to the quaternary system (Figure 6 Panel-C). These domains coalesce quickly before a surface

15

pressure of 15 mN/m is reached. Upon the addition of gelatine NPs, LC domains appear at a

16

surface pressure of 10 mN/m but quickly coalesce into a film at surface pressures of 12 mN/m

17

(Figure 6 Panel-D). At a surface pressure of 30 mN/m, the film void structures are observed and

18

are indicated by an arrow and a box (zoomed-in insets) in Figure 6 (Panel-D).

19


22

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1 2 3 4 5 6

Figure 6. BAM images of Quaternary of molar ratio of 45 DPPC: 8 POPC: 1 DPPG: 6 POPG (A), Quaternary + NPs (B), Quinary with 2% total weight cholesterol (C), Quinary + NPs (D). The ratio of lipids to NPs is 10:1 w/w. The arrow shows to area which is magnified (2X) and displayed in the insert of panel (D) which indicate the presence of film voids. Figure insets in (D) refers to film defects. n≥2

7 8 9 10

3.3.4 Domain Size Analysis

11

A quantitative analysis of the domain sizes is presented in Figure 7. Since domain formation

12

occurred at different pressures, size should not be compared quantitatively across systems. The

13

formation of domains may be induced by the presence of NPs and size may be affected. We

14

cannot assume that only line tension is responsible for the size differences.


23


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Page 24 of 36

1

However, changes in the domain size in each system before and after addition of NPs are

2

reported. For the DPPC system, it is observed that the addition of NPs significantly reduces the

3

diameter of the observed domains from 15 µm to about 7.5 µm. A similar trend is observed for

4

the binary PC system whereby the NPs reduce the domain diameter from about 14 µm to 8 µm.

5

This could be due to preferential interactions between the gelatine NPs and the rigid saturated PC

6

species as seen in the BAM images in (Figure 4 Panel F). On the other hand, the binary PG

7

system shows only a slight decrease of about 1 µm in the domain diameter when compared to

8

binary PC. These results support a mechanism of interaction that is dependent on the lipid head

9

group characteristics. In addition, lipid-NP clusters are found at the edges of LE-LC phases. The

10

PGs have a negatively charged head group while the gelatine NPs have a positively charged

11

surface. The overall surface charge density will provide attractive electrostatic forces across the

12

entire monolayer. Since the LE components occupy larger areas, it is not unexpected to see more

13

NP clusters in the fluid areas of the film.

14

For the more complex systems, the domains are analyzed at lower pressures (10-12 mN/m)

15

where domains are observed in the controls. Addition of NPs increased the domain sizes from

16

4.5 to 6 µm for the quaternary system and from 2.5 to 3 µm for the quinary system. This also

17

supports the overall trends discussed before that NPs induce rigidity in all systems, since

18

domains in the complex systems were only observed in the presence of NPs. Nevertheless, these

19

domains are smaller and less frequent than in the binary systems indicating the complex packing

20

structure and reduced surface charge density allow better adaptation to the presence of the NPs.


24

Page 25 of 36

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1 2 3 4 5 6 7 8 9

Figure 7. Average domain diameter for different lipid systems at different surface pressures. For the DPPC and DPPC+NPs systems, the diameters are measured at surface pressures of 7 and 10 mN/m respectively; for the binary PC and PG systems, the diameter was measured at a surface pressure of 15 and 30 mN/m, respectively; for the quaternary and quinary systems the diameter were measured at 15 and 10 mN/m, respectively. These surface pressures are system specific. Black bars: pure lipid systems, grey bars: lipid system + NPs. The differences in the domain sizes within a system are confirmed to be significant by a t-test: * significant at p < 0.05, **** significant at p < 0.0001

10 11

3.3.5 3D Visualization of DPPC

12

Figure 8 shows the 3D visualization of the DPPC system with and without the NPs. As

13

mentioned above, the 3D image is a qualitative way of analyzing changes in the monolayer

14

thickness based on the relative peak intensities. No bright clusters are seen in DPPC without NPs

15

(Figure 4 Panel A and B). These structures indicate domains that are protruding from the

16

monolayer film and start forming at low pressures of 10 mN/m. As well as increasing in

17

frequency and intensity with the surface pressure upon compression. Similar peaks have been

18

reported by our group for DPPC and gelatine NPs39 but were also observed for the hydrophobic


25


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Page 26 of 36

1

transmembrane protein EmrE and selected bacterial lipid classes46 as well for complex bacterial

2

extracts72.

3

The observed lateral organization visualized by BAM is altered upon NP addition despite the fact

4

that overlapping isotherms are exhibited by these systems. Minimal effects of particles on

5

isotherms were also reported by Tatur and Badia for 0.2 mol % gold NPs on the major LS

6

component DPPC as well as on the clinically used lung surfactant replacement, Survanta78.

7

Similarly as reported here, a significant change of the lateral organization of the monolayer was

8

observed whereby the domains induced by NPs addition were smaller and appeared in greater

9

numbers78. Guzmán et al. also reported that hydrophilic silica NPs did not change the isotherms

10

of binary lipid monolayer systems whereas BAM images demonstrated that the lateral

11

organization was noticeably changed79. The observed BAM images show that the addition of

12

silica NPs prevented the formation of the domains observed for the pure binary system up to a

13

surface pressure of 40 mN/m, thus favouring well mixed lipid phases79. These papers confirm the

14

trends of the presented results. Future studies of the effect of multiple compression cycles on

15

monolayer stability and lateral film structure will provide further insight on the significance of

16

this reorganization.

17

Nevertheless, these data already document preferential interactions of nanoparticles with specific

18

lipid components leading to partial lipid demixing and cluster formation. Even small changes

19

caused by the presence of nanoparticles could impair proper lung function over time causing

20

significant health problems such as acute respiratory distress syndrome80.


26

Page 27 of 36

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1 2 3 4 5

Figure 8. BAM images and 3D representation of DPPC. BAM image of pure DPPC at a surface pressure of 7 mN/m (A), 3D representation of pure DPPC at 7 mN/m (B), BAM image of DPPC + NPs at a surface pressure of 10 mN/m (C), and 3D representation of DPPC + NPs system at 10 mN/m.

6 7

In addition, it has to be emphasized that this approach is useful in screening different

8

formulations of nanoparticle based drug carriers before their administration into the body. It was

9

demonstrated that nanoparticles coated to minimize detection by the immune system,

10

significantly reduced the collapse pressure in-vitro. This effect was directly correlated to the in-


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

vivo outcome; animals exposed to uncoated NPs were not affected whereas animals exposed to

2

the coated particles did not survive treatment81.

Page 28 of 36

3 4

5. CONCLUSIONS

5

The addition of gelatine NPs in a 10:1 lipid to NP ratio does not cause major changes in the

6

compression pressure-area isotherms. Nonetheless, a significant impact on the lateral

7

organization of the biomimetic films upon addition of gelatine NPs is evident from the BAM

8

results.

9

The presence of monounsaturated side chains has a strong impact on lipid-NP interactions as

10

seen for the lateral reorganization of POPC films. Furthermore, the data suggest electrostatic

11

interactions between the positively charged gelatine NPs and negatively charged PGs. The slight

12

shift of the isotherm to lower areas upon NP addition to the POPG system is supported by the

13

BAM images of the clusters that appear throughout the monolayer indicating tighter packing

14

presumably due to strong electrostatic interactions when compared to POPC. Charge density

15

may be relevant as tighter packed saturated DPPG showed much stronger effects in lateral film

16

organization. In the presence of NPs, bright clusters protruding from the film are observed. More

17

complex systems are able to better adapt to the presence of NPs indicated by a substantial

18

reduction in cluster formation.

19

Therefore, several factors such as lipid head group structure, side chain architecture as well as

20

charge and charge distributions that also depend on the molar ratio of the mixtures affect lipid-


28

Page 29 of 36

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1

NP interactions. Even components present at low concentrations (such as DPPG and POPC)

2

clearly have an impact on the lateral organization of the films.

3

The biophysical study identified key factors affecting the interactions of NPs with complex

4

biomimetic LS models. These factors are relevant for the development of novel drug carrier but

5

have not been sufficiently considered so far. In addition, complex but well defined models

6

improve the ability to test and eventually predict the toxicity of various nanomaterials in an

7

important effort to reduce nanotoxicity.

8 9

AUTHOR INFORMATION

10

Corresponding Author

11

* email: [email protected]

12

Author Contributions

13

The manuscript was written through contributions of all authors. All authors have given approval

14

to the final version of the manuscript.

15

ACKNOWLEDGMENT

16

The work was supported by a DG grant from NSERC. WD is supported by a QE II scholarship.

17

ABBREVIATIONS

18

LS,

19

Palmitoyloleoylphoshphatidylglycerol; SP-A,B,C,D, Surfactant Protein A,B,C,D; NP(s),

20

Nanoparticle(s); BAM, Brewster angle microscopy; PC(s), Phosphatidylcholine(s); PG(s),

Lung

Surfactant;

DPPC,

Dipalmitoylphosphatidylcholine;

POPG,


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Phosphatidylglycerol(s);

POPC,

Palmitoyloleoylphosphatidylcholine;

2

Dipalmitoylphosphatidylglycerol; LE, Liquid Expanded; LC, Liquid Condensed

Page 30 of 36

DPPG,

3 4 5 6 7 8 9

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