Effect of Cationic Surfactants on the Molecular Organization of the Simplified Model Lipid Envelope of SARS-CoV‑2 VirusInsights from the Langmuir Monolayer and Liposome Studies

Abstract

In this study, we compare the physicochemical interactions of cationic surfactants, cetyltrimethylammonium bromide (CTAB) and cetylpyridinium chloride (CPC), at concentrations lower than their CMC with model systems mimicking the lipidic content of the SARS-CoV-2 envelope in terms of the charge of polar heads and the composition of acyl chains. The DOPC/DMPS/PI 50:35:15 (molar ratio) Langmuir monolayers treated as 2D models exhibited increased fluidity and a shift of surface pressure–area per molecule isotherms toward larger areas upon surfactant interaction. Liposomes (3D models) showed an increase in hydrodynamic diameter and changes in zeta potential in the presence of CTAB and CPC. These effects were observed both in water and PBS buffer (pH 7.4), while in PBS the changes were more pronounced in the monolayer systems and surfactant-dependent for liposomes. Additionally, giant unilamellar vesicles (GUVs) were prepared and visualized using fluorescence microscopy to follow shape fluctuations and rapid changes in fluorescence intensity upon exposure to surfactants. Despite the electrostatic attractions governing the lipid envelope-surfactant interactions, the differences in the interaction mechanisms arise from the molecular structure of the polar headgroups of surfactants: CPC is more likely to effectively penetrate into the lipid layers due to its planar, aromatic headgroup, while CTAB’s bulky polar head leads to its accumulation close to the lipid surface. These findings provide new insights into molecular-level interactions between low-concentration cationic surfactants and more exact models of viral lipid envelopes and may serve as a basis for the development of mutation-independent antiviral strategies targeting the lipid components of enveloped viruses.

Introduction

Enveloped viruses are small particles with a simple structure that exhibit an ability to infect organisms. They are built of a lipid envelope that protects a protein capsid containing the viral genetic material. The main function of a lipid envelope is to increase the virus’s resistance to external factors. Therefore, changing its physicochemical properties could lead to a weakening of the virus or even its complete disruption. , Only a small number of enveloped viruses have been studied in terms of the lipid composition of their envelope, although available data indicate that the main components include phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and sphingomyelin (SM), as well as cholesterol and viral proteins. −

SARS-CoV-2 is a coronavirus belonging to a class of enveloped viruses that can be transmitted very quickly through droplets or contact with contaminated objects. In March 2020, it spread worldwide, causing a global pandemic called by WHO. It is built of a single-stranded RNA, a lipid envelope, a protein capsid, and viral proteins: nucleocapsid protein (N), spike protein (S), membrane protein (M), and envelope protein (E). Coronaviruses show a high tendency for genetic recombination and mutations, leading to new strains that are often resistant to previously effective antiviral treatments, which are based on highly selective drugs that target viral proteins essential for replication. The progressing resistance of SARS-CoV-2 virus creates a need for a deeper understanding of the physicochemical properties of its lipid envelope, which is characterized by an increased amount of negatively charged lipids, including phosphatidylinositol (PI) and phosphatidylserine (PS). The negatively charged viral lipid envelope can be treated as a potential target for selected cationic compounds such as, e.g., cationic surfactants, since envelope disintegration resulting from the exposure could lead to the reduction of the virus infectivity.

Surfactants are amphiphilic molecules that can modify the physicochemical properties of interfaces and self-associate into micelles at concentrations above their critical micelle concentration (CMC). Their structure consists of a hydrophilic polar headgroup and a hydrophobic alkyl chain, and they may exist in ionic or nonionic forms. , Depending on their concentration relative to the CMC, the alkyl tail length, and the presence of an electric charge, surfactants can change the properties of model membranes. In liposomes, they can affect their size and shape through incorporation into the lipid bilayer or may induce loss of bilayer integrity and solubilization as a result of flip-flop, which enables interleaflet translocation of nonionic surfactants (for ionic surfactant, it is significantly less efficient). − Liposome solubilization is described by a three-stage model starting from the adsorption of surfactant molecules at the membrane surface up to the formation of mixed micelles. There are also surfactants whose effects on liposomes are independent of the applied concentration due to the composition of their alkyl chains. Gooran et al. discuss two mechanisms of lipid bilayer solubilization differing in their dependence on the CMC and in the reversibility of the resulting defects. In another study, it was shown based on molecular dynamics (MD) simulations that in the case of the SARS virus, ionic surfactants mostly adsorb onto the surface of the spike protein, causing its collapse and deformation of the lipid envelope. The following experimental studies on coronaviruses confirmed this mechanism for ionic surfactants. In contrast, nonionic surfactants are more strongly directed toward solubilization of lipid bilayers. A comprehensive description of the mechanisms by which surfactants act on viral envelopes was provided by Negi et al. Three interaction pathways were identified depending on surfactant charge: deformation of the bilayer because of asymmetric incorporation of cationic surfactants, solubilization of viral proteins by anionic surfactants, and envelope swelling as a consequence of flip-flop of nonionic surfactants.

To fully investigate the selectivity, transport mechanisms, and interactions of surfactant molecules with lipid membranes mimicking models of viral lipid envelopes, several complementary methods should be combined. Liposomes should be studied due to their spherical shape, the presence of a lipid bilayer, and membrane curvature, which make them structurally similar to real virions surrounded by the envelope. To support liposome studies, planar Langmuir monolayers at the air–water interface were also employed. The Langmuir method makes it easier to study lipid interactions under controlled conditions and allows for a better understanding of the mechanisms without additional effects such as membrane curvature or the presence of multilayers. It also allows for the simplification of a highly complex structure to single- or multicomponent monolayers with specific composition and condensation. , Due to this simplicity, the mechanisms of such interactions can be better understood and described. Sęk et al. investigated the impact of ionic surfactants on model lipid membranes, describing their penetration process, the influence of surfactant charge and alkyl chain length, and the presence of cholesterol within the lipid layer using both planar and spherical lipid models. ,

In this work, we present for the first time a comprehensive physicochemical characterization of the interactions of two cationic surfactants with identical alkyl-chain lengths: cetylpyridinium chloride (CPC) and cetyltrimethylammonium bromide (CTAB) (Figure ) with simple models of viral lipid envelopes. The choice of the surfactant molecules was motivated by their widespread application in disinfecting and antiviral agents, including mouthwashes, oral rinses, throat and nasal sprays, as well as surface disinfection agents. Each surfactant was applied at two concentrations lower than their respective critical micelle concentrations (CMCs) in order to follow more discrete changes in the physicochemical properties of the model viral envelopes before their complete disintegration when exposed to the above-CMC concentrations of surfactants. The impact of the selected surfactants on two- and three-dimensional models of the SARS-CoV-2 lipid envelope prepared in both water and PBS buffer environments was determined. Such a comprehensive approach allowed us to explicitly show the importance of electrostatic interactions, as well as the effect of the geometry of the surfactant polar headgroup and the influence of the environment (i.e., the presence or absence of ions in the buffer or pure water, respectively). These aspects strongly affecting the surfactant-lipid interactions have not been addressed systematically so far. The lipid models were prepared as ternary mixtures with a composition carefully selected to more precisely reflect the actual phospholipid composition of the lipid envelope of the SARS-CoV-2 virus in terms of both composition and charge of polar heads as well as acyl chain length and saturation, focusing mostly on potential electrostatic interactions with selected surfactants. This approach has not been employed so far in the studies of surfactant-lipid interactions. The results reported in this paper advance our physicochemical understanding of the mechanisms of the interactions and possible destabilization of the lipid envelope of SARS-CoV-2 by low concentrations of cationic surfactants.

1.

Structures of (A) DOPC, (B) DMPS, (C) Soy PI (main component of the extract), (D) CPC, and (E) CTAB.

Experimental Section

Materials

Cetylpyridinium chloride (CPC) and cetyltrimethylammonium bromide (CTAB) were purchased from Merck. Phospholipids, including 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (sodium salt) (DMPS), and l-α-phosphatidylinositol (sodium salt) (Soy extract) (PI) (Figure ), were purchased from Avanti Polar Lipids (USA) and used without further purification. Stock solutions of lipids were prepared in a chloroform/methanol 4:1 v/v mixture (HPLC grade, Merck) to achieve the final concentration of 0.5–1 mg/mL, which were then mixed to obtain the DOPC/DMPS/PI 50:35:15 molar ratio mixture to mimic the SARS-CoV-2 lipid envelope. Phosphate-buffered saline (PBS) solution was prepared by dissolving sodium chloride, potassium chloride, disodium phosphate, and monosodium phosphate in MiliQ water (resistivity of 18.2 MΩ cm) to obtain a final salt concentration of 0.01 mol/L and by titrating it using 0.1 mol/L HCl and 0.1 mol/L NaOH to adjust the final pH to 7.4.

Surface PressureArea per Molecule Isotherms

The surface pressurearea per molecule measurements were carried out using a KSV Nima Langmuir trough of dimensions 78.4 × 74 cm (580 cm²) equipped with two hydrophilic barriers and a Wilhelmy microbalance employing a piece of filter paper to measure the surface pressure. Before the experiment, the Langmuir trough was cleaned with methanol and chloroform, followed by extensive rinsing with Milli-Q water. The experiments began by filling the trough with either pure water or PBS buffer, or solutions of surfactantscetylpyridinium chloride (CPC) and cetyltrimethylammonium bromide (CTAB)at concentrations of 10^–5 mol/L in water and 10^–6 mol/L in buffer. A lipid mixture solution was then deposited onto the subphase surface using a Hamilton microsyringe and left for 10 min for solvent evaporation. During the measurement, the barriers were moving symmetrically at a constant speed of 10 mm/min, and surface pressure changes were observed on the surface pressure–area isotherm. All measurements were carried out at 21 °C and were repeated at least twice.

From the recorded isotherms, the compression modulus (C _s ) was calculated as the reciprocal of monolayer compressibility ,

$$C_{\mathrm{s}}^{-1}=-A{\left(\frac{\mathrm{d}\pi }{\mathrm{d}A}\right)}_T$$ 1

where C _s is the compression modulus [mN/m], A is the area per molecule [Å], and π is the surface pressure [mN/m]. The values of the compression modulus provide insights into the elasticity and stability of the monolayer. These values were used to construct C _s –π plots, in which local minima indicate phase transitions or partial collapse, while the maximum corresponds to achieving the maximum packing density of molecules. Based on C _s maximum values, the monolayer phase states can be defined as follows: C _s in the range of 0–12.5 mN/m indicates a gas phase (G), 12.5–50 mN/m indicates a liquid-expanded phase (LE), 100–250 mN/m indicates a liquid-condensed phase (LC), and a solid phase (S) is expected for values exceeding 250 mN/m.

Electrical Surface PotentialArea per Molecule Isotherms

The surface potential was measured simultaneously with the surface pressure using a surface potential sensor (SPOT, KSV Nima). The reference electrode was positioned at the bottom of the trough, and the vibrating plate was placed approximately 2 mm above the subphase surface. Prior to each measurement, both elements were thoroughly cleaned with methanol, and the value of surface potential was zeroed after filling the trough with the subphase. The relationship between surface potential and the molecular area for monolayers carrying a nonzero net charge is described by the following equation

$$\mathrm{\Delta }V=\frac{{\mu }_{\mathrm{z}}}{\epsilon }·\frac{1}{A}+{\mathrm{\Psi }}_{\mathrm{D}\mathrm{L}}$$ 2

where ΔV is the surface potential, μ_z is the component of the molecular dipole moment perpendicular to the subphase surface, ε is the permittivity of the medium, A is the area per molecule, and Ψ_DL is the double-layer potential. For the recorded surface potential–area per molecule (ΔV–A) isotherms, the maximum surface potential values (ΔV _max) corresponding to the most vertical orientation of the phospholipids at the air–water interface were also determined. Each measurement was repeated at least three times to ensure reproducibility.

Liposome Formation

Three-dimensional bilayer lipid models were prepared following the Bangham method. Single-component lipid stock solutions (5 mg/mL) were prepared in a chloroform/methanol mixture (4:1, v/v) and were combined in appropriate proportions to obtain a ternary lipid mixture DOPC/DMPS/PI in a molar ratio of 50:35:15. Volumes of 100 μL were dispensed into Eppendorf tubes and secured with parafilm to limit oxygen access and protect the unsaturated alkyl chains from oxidation. The mixtures were vortexed, and the solvent was evaporated under a stream of argon to obtain a dry film. These films were then placed in a vacuum desiccator for 2 h, followed by storage at −4 °C. 24 h prior to the measurement, 0.5 mL of PBS buffer (pH 7.4) was added to a tube, and the sample was subjected to ultrasonication for 1 h and 45 min at 37 °C - a temperature selected to exceed the phase transition temperatures of the constituent lipids. , The hydrated lipid films were then stored at 3 °C for at least 24 h to stabilize the liposomes prior to further studies.

Hydrodynamic Diameter and Zeta Potential Measurement of Liposomes

Liposome characterization was carried out using a Zetasizer Nano ZSP (Malvern Panalytical, Malvern, UK) operating at a fixed scattering angle of 173° with data acquisition via Malvern software. Calculations were based on the Helmholtz–Smoluchowski equation. The hydrodynamic diameter (d) distribution and zeta potential (ζ) were measured for liposome solutions with lipid concentrations of 0.05 and 0.0025 mg/mL, respectively, diluted in either Milli-Q water or PBS buffer (pH 7.4) and placed in a quartz cuvette or a dedicated zeta potential cuvette (Malvern Panalytical, Malvern, UK). Prior to each measurement, samples were mixed by hand for 1 min and then thermostated at 25 °C for 2 min to ensure sample equilibration. Measurements were recorded continuously over a 120 min period at constant temperature. Each measurement was repeated at least three times.

Giant Unilamellar VesiclesFormation and Visualization

Giant unilamellar vesicles (GUVs) were prepared using the electroformation method. Initially, a ternary lipid mixture was prepared by combining lipid stock solutions (10 mg/mL each in a chloroform/methanol mixture (4:1, v/v)) in a molar ratio of DOPC/DMPS/PI 50:35:15, supplemented with 1 mol % of the fluorescent probe NBD-PC (PC probe, Avanti Polar Lipids, USA). The lipid mixture was diluted with chloroform and deposited in a Parafilm-sealed, homemade electroformation chamber containing two platinum electrodes. The organic solvents were evaporated under a gentle stream of argon, and the obtained lipid film was further dried in a vacuum desiccator for 2 h. Subsequently, the chamber was filled with a 200 mmol/L sucrose solution prepared in PBS buffer (pH 7.4), and an alternating current electric field (10 Hz, 1.0 V amplitude) was applied for 1.5 h to promote vesicle formation. For imaging, GUV suspensions were diluted to a lipid concentration of 0.0075 mg/mL in a 200 mmol/L glucose solution prepared in PBS (pH 7.4). The samples were transferred to Millicell EZ SLIDE 8-well glass chambers (Merck KGaA, Darmstadt, Germany) and observed at room temperature using a fluorescence microscope (Nikon Eclipse Ni-U, Tokyo, Japan) over a 30 min period. Surfactants were introduced at a final concentration of 10^–5 mol/L (below CMC) and 10^–3 mol/L (above CMC). Images were acquired using JENOPTIK GRYPHAX software, and each imaging series was repeated three times to ensure reproducibility.

Results and Discussion

Surface Pressure and Surface Potential Measurements

Ternary Langmuir monolayers formed at the air–water interface were examined as 2D models of the SARS-CoV-2 lipid envelope. The isotherm referring to DOPC/DMPS/PI monolayer compression on the water subphase is similar to the one presented previously in the same conditions. Both exhibit a lift-off around 100 Ų, a partial collapse of the monolayer at higher surface pressures, and an LC state of the monolayer (Figure A).

2.

(A) Surface pressure–area per molecule isotherms (π–A) and (B) surface potential–area per molecule (ΔV–A) isotherms of DOPC/DMPS/PI 50:35:15 monolayers formed on pure water (black) and water containing 10^–5 mol/L CTAB (red) and 10^–5 mol/L CPC (blue). Inset: compression modulus versus surface pressure (C _s ^–1–π) plots.

The presence of low concentrations, well below CMC (Table S1) of CTAB and CPC, in the water subphase causes changes in the organization and fluidity of the DOPC/DMPS/PI monolayer. The isotherms are shifted toward larger areas for both surfactants (Figure A). The values of C _s ^–1 _max decrease to 89 and 77 mN/m for subphases containing CTAB and CPC, respectively (Table ). Thus, the monolayers are in the liquid-expanded state, indicating lower organization of lipid molecules in the presence of surfactants. CTAB causes an increase in partial collapse surface pressure, suggesting an enhanced stability of the layer and a change in its shape that suggests a two-step exclusion of DOPC and SM from the monolayer upon surfactant interactions with lipids. It could indicate specific interactions of CTAB with one of the components or the ability to affect lipid domains in a specific manner. On the other hand, CPC causes a decrease in partial collapse surface pressure, which starts at the point where the isotherm for the CPC-containing system reaches the same area per molecule as the isotherm of the surfactant-free system at the surface pressure of 40 mN/m. It may suggest an exclusion of CPC molecules as a result of compression or lipid loss as a consequence of CPC-lipid interactions, which leads to the decreased stability of the ternary monolayer. These results demonstrate that both surfactants affect lipid packing and the physicochemical properties of the compressed monolayer in a surfactant-specific manner, which may depend on the length of the hydrocarbon chain, the composition of the polar headgroup, and electric charge. , Both surfactants are cationic, which suggests the presence of attractive electrostatic interactions with negatively charged phospholipids of the ternary mixture: DMPS and PI. Although the alkyl chains of the surfactants have identical lengths, the pyridinium aromatic moiety in CPC exhibits a more planar configuration of polar headgroup compared to the trimethylammonium group of CTAB (Figure ). Therefore, the differences in the shape of the isotherms may indicate different mechanisms of action on the monolayer, including the more efficient destabilization of the monolayer leading to the collapse at lower surface pressures induced by CPC compared to CTAB.

1. Characteristic Parameters of the Langmuir Monolayers of the DOPC/DMPS/PI 50:35:15 Mixture Used as a 2D Model of the Viral Lipid Envelope of SARS-CoV-2 Formed on the Water Subphase and Water Subphase-Containing Surfactants.

Subphase	A π=10 mN/m [Å2]	A π=30 mN/m[Å2]	A coll [Å2]	πcoll [mN·m–1]	C s –1 max [mN· m–1]	A c [Å2]	ΔV [V]
H2O	82.4 ± 1.2	62.4 ± 1.2	51.9 ± 0.9	47.5 ± 0.3	117 ± 3	223.8 ± 5.7	0.265 ± 0.061
10–5 mol/L CTAB (H2O)	123.3 ± 5.0	87.9 ± 2.9	71.0 ± 2.2	47.8 ± 1.1	89 ± 2	204.5 ± 5.4	0.383 ± 0.029
10–5 mol/L CPC (H2O)	105 ± 2.5	68.9 ± 3.8	59.2 ± 2.8	40.0 ± 0.9	77 ± 3	277.5 ± 14.6	0.350 ± 0.010
PBS buffer	90.3 ± 6.2	65.8 ± 6.4	56.2 ± 5.8	41.8 ± 1.3	91 ± 16	206.9 ± 4.7	0.338 ± 0.017
10–6 mol/L CTAB (PBS)	174.6 ± 9.8	85.0 ± 7.7	57.1 ± 6.5	47.4 ± 0.2	53 ± 3	-	0.443 ± 0.016
10–6 mol/L CPC (PBS)	208.7 ± 15.1	106.8 ± 3.1	83.1 ± 1.9	39.9 ± 1.8	56 ± 14	-	0.414 ± 0.055

To gain knowledge about the electrical properties of the analyzed lipid monolayers, changes in surface potential were measured and shown as surface potential–area per molecule isotherms (ΔV–A) (Figure B). Measurements of surface potential were started at large areas per molecule before reaching the critical area (A _c), at which the components of the monolayer start to reorient and surface potential starts to increase. The maximum surface potential ΔV _max was determined as the point corresponding to the maximum value of the compressibility modulus C _s ^–1 _max, as ΔV _max corresponds to the most vertical orientation of the phospholipids at the air–water interface. For the lipid layer formed on a water subphase, the critical area A _c was approximately 225 Ų/molecule, and ΔV _max reached 0.265 V (Table ). The ΔV–A curve showed two distinct slopes, with a transition around 160 Ų/molecule. The corresponding surface pressure remains near zero; therefore, the observed change in μ _z (eq ) indicates the gas–liquid expanded phase transition. The critical molecular areas and maximum surface potential in the presence of CTAB and CPC were 205 Ų and 278 Ų and 0.383 V and 0.350 V, respectively (Table ). Thus, at the beginning, both surfactants influence dipole orientation at the interface in opposite ways because of differences in the composition of their polar headgroups; CTAB hinders the reorientation, whereas CPC promotes it. Beyond these points, a sharp increase in surface potential is observed, referring to electric double layer formation. This is likely related to the accumulation of positively charged surfactant molecules close to the interface, where negatively charged polar headgroups of lipids are adsorbed.

To estimate the effect of counterions on the interactions, a 2D model of the SARS-CoV-2 lipid envelope was also formed on the PBS subphase (pH 7.4). The DOPC/DMPS/PI mixture forms stable monolayers on the pure PBS subphase in a liquid-expanded (LE) phase, as evidenced by C _s ^–1 _max value reaching approximately 90 mN/m (Table ). Compared to the water subphase system, the monolayer is less organized in the presence of counterions interacting with polar headgroups of phospholipids. At surface pressure values around 40 mN/m, there is a plateau corresponding to a partial collapse of DOPC and PI components, which is in agreement with C _s ^–1 values reaching 0 above π = 45 mN/m in C _s ^–11–π plot (Figure A).

3.

(A) Surface pressure–area per molecule isotherms (π–A) and (B) surface potential–area per molecule (ΔV–A) isotherms of DOPC/DMPS/PI 50:35:15 monolayers formed on the PBS subphase (gray) and PBS subphase-containing 10^–6 mol/L CTAB (light red) and 10^–6 mol/L CPC (light blue). Inset: compression modulus versus surface pressure (C _s ^–11–π) plots.

The influence of both surfactants on the DOPC/DMPS/PI monolayer formed on the PBS buffer is similar (Figure ). Despite the use of a concentration of 10^–6 mol/L, which is 1 order of magnitude lower than in pure water to keep it well below their CMCs in buffer (Table S1), some of the surfactant molecules are adsorbed at the air-buffer interface before starting the compression, which leads to a shift of isotherms toward larger areas compared to the pure water subphase. It happens because of the increased surface activity of surfactants in the buffer and their contribution to the layer at the air-buffer interface. Additionally, they may form relatively stable monolayers when dissolved in an organic solvent and spread directly at the air-PBS buffer interface (Figure S1). The presence of counterions in the subphase leads to changes in lipid–lipid, lipid-surfactant, and surfactant–surfactant interactions. , It is also manifested by lower maximum values of compression modulus C _s ^–11 _max , which decrease to 53 and 56 mN/m for buffered subphases containing CTAB and CPC, respectively (Table ). However, these effects are more difficult to observe by the Langmuir technique alone, as the presence of counterions may stimulate the formation of micelles formed from cationic surfactants by reducing the strength of their mutual electrostatic repulsions. , It is also worth noting that CTAB causes an increase in the partial collapse surface pressure, and, unlike in water systems, it did not affect the exclusion process of both DOPC and PI molecules at the same time, which suggests a stabilizing effect of CTAB on the lipid monolayer in the presence of counterions. However, CPC interactions with lipids lead to a decrease in the surface pressure of the partial collapse, similarly to water systems and to a prolonged exclusion process of DOPC and PI molecules. This indicates that CPC affects the organization and exclusion processes within the lipid layer to a greater extent than does CTAB.

Surface potential-area per molecule isotherms (ΔV–A) were also recorded for those systems. However, due to their complexity, they are analyzed only in general. The critical molecular area A _c is 207 Ų, and the maximum surface potential ΔV reaches 0.338 V (Table ). The ΔV–A curve indicates a gradual reorientation of the molecules at the interface. In the presence of surfactants, it was not possible to determine the critical area A _c, as the surface potential was already above zero after spreading the lipids and solvent evaporation (Figure B). Probably the adsorption of surfactant molecules at the interface and the presence of PBS buffer counterions enhanced mutual attractive lipid–surfactant interactions. The slope of the ΔV–A isotherm in the absence and presence of CPC is comparable, but the isotherm for the CTAB-containing system exhibits a change in slope around 160 Ų, indicating molecular reorganization at the interface.

2. Characteristic Parameters of DOPC/DMPS/PI 50:35:15 Liposomes Used as a 3D Model of the Viral Lipid Envelope of SARS-CoV-2.

	0 min		60 min		120 min
	d [nm]	ζ [mV]	d [nm]	ζ [mV]	d [nm]	ζ [mV]
H2O	118 ± 8	–50 ± 2	106 ± 11	–51 ± 4	120 ± 8	–43 ± 6
10–5 mol/L CTAB	176 ± 8	–40 ± 5	137 ± 2	–48 ± 2	156 ± 8	–42 ± 6
10–5 mol/L CPC	140 ± 8	15 ± 2	136 ± 9	–8 ± 2	144 ± 11	–13 ± 2
PBS	85 ± 6	–20 ± 1	99 ± 10	–17 ± 1	103 ± 2	–20 ± 3
10–5 mol/L CTAB	153 ± 1	–9 ± 4	142 ± 8	–15 ± 2	123 ± 5	–17 ± 1
10–6 mol/L CPC	193 ± 6	–22 ± 0	218 ± 7	–23 ± 1	195 ± 6	–22 ± 1

Size and Zeta Potential of Liposomes

Liposomes composed of DOPC/DMPS/PI 50:35:15 mixture and prepared according to the Bangham method (Section 2.2.3) served as 3D models of the SARS-CoV-2 lipid envelope. Liposomes tested in pure water are characterized by a hydrodynamic diameter of about 120 nm (Table ) and remain stable over time (Figure A,B). Their size corresponds to the actual size of SARS-CoV-2 viral particles, which is in the range of 80–130 nm and is consistent with previous results. However, zeta potential values of liposomes measured in pure water changed slightly over time (Figure C,D and Table ), which can be explained by the repulsive electrostatic interactions between negatively charged lipids, leading to their reorganization.

4.

Size distribution of particles in pure water (black), water containing 10^–5 mol/L CTAB (red), and water containing 10^–5 mol/L CPC (blue) measured by DLS (A) at the beginning of the experiment, (B) 120 min after the start of the experiment, (C) changes of particle size over time, and (D) changes of zeta potential over time.

The addition of 10^–5 mol/L CTAB caused a change in particle size distribution over time (Figure and Table ). The trend is not linear, indicating a reorganization of the lipids in the bilayer as a result of surfactant-lipid interactions. Similarly, the presence of CPC also caused changes in the particle size distribution but to a smaller extent. In both cases, size distributions did not change significantly after 120 min, but under the influence of CTAB, the size fluctuations of liposomes are higher compared to CPC.

The addition of CTAB (10^–5 mol/L) led to a slight increase in zeta potential to −40 mV at the beginning, followed by the decrease. This trend is similar to liposomes in water until both systems become similar after 60 min (Figure D). It suggests that the lipid headgroup’s negative charge is first screened by positive charges coming from CTAB, which confirms the previously observed surfactant-lipid interactions and accumulation of surfactant molecules near the surface of liposomes. Later, CTAB molecules are removed from the area around the liposomes, and a state of equilibrium is reached. However, the changes in zeta potential and hydrodynamic diameter are quite different over time. Probably after an initial strong binding, some surfactant molecules that interact weakly with lipids may partially detach and remain in the vicinity of the liposomes. As a result, the zeta potential values are close to those of the original liposomes, even though the particle size remains affected by the initial interactions. On the other hand, the presence of CPC molecules (10^–5 mol/L) caused a significant increase in zeta potential values to +15 mV at the beginning with the tendency to reduce its value in time. It suggests the formation of structures with a high-density of positive charge on the surface, which could be CPC aggregates formed based on the interactions between the electron cloud coming from double bonds and the nitrogen cation of pyridine. , It could mean that a greater number of CPC molecules incorporate into the lipid bilayer and accumulate close to its surface, increasing their hydrodynamic diameter and screening negative surface charge, which is consistent with previous results. ,

To compare the observed differences in more detail, it is necessary to closely analyze the structure of surfactant polar headgroups. CPC’s headgroup is planar aromatic pyridine, while the CTAB headgroup is built of a bulky trimethylammonium cation (Figure ). Both headgroups differ also in their sizes, which may influence their ability to incorporate into closely packed phospholipid headgroups of the outer leaflet. The importance of the differences in the headgroup size can be justified using thermodynamic analysis of the incorporation process. The energetic cost of transferring a monovalent ion of r radius (in this case, we consider the polar headgroup of a surfactant) from water solution into a lipid bilayer can be calculated using the process’ free energy

$$\frac{\mathrm{\Delta }G}{kT}=\frac{{\mathcal{l}}_{\mathrm{b}}^{\mathrm{o}}}{2r}(\frac{1}{{\epsilon }_{\mathrm{b}}}-\frac{1}{{\epsilon }_{\mathrm{w}}})$$ 3

where k is the Boltzmann constant, T is the temperature of the experiment, ${\mathcal{l}}_{\mathrm{b}}^{\mathrm{o}}$ = 560 Å is Bjerrum length in a vacuum, ε_b = 2 is the electrical constant for a bilayer, and ε_w = 80 is the electrical constant for water. CTAB and CPC polar headgroup radius values used are 1.064 and 2.458 Å, respectively, based on the calculations made in the Avogadro program. It turns out that ΔG of transferring CTAB’s polar headgroup within a lipid bilayer would reach 74.9 kcal/mol, while for CPC, this value equals 32.4 kcal/mol. Obtained values suggest that it would be very energetically demanding for CPC and CTAB molecules to penetrate both leaflets of the lipid bilayer. Nevertheless, transferring the CPC molecule is less demanding energetically compared to CTAB, especially considering its planar structure. , Therefore, it may be assumed that CTAB molecules will mostly tend to accumulate close to the surface of the liposomal bilayer, whereas CPC molecules may incorporate within the lipid bilayer, at least to a greater extent than CTAB. The driving force of these processes is the attractive electrostatic interactions between the cationic surfactants and the negatively charged phospholipids.

The observed surfactant-liposome interactions are different when pure water is exchanged with PBS buffer (Figure ). Histograms indicate the tendency of liposomes in pure PBS to increase their hydrodynamic diameter distribution within 120 min (Figure A,B), but their values are smaller compared to water (Table ). It is due to the reduced repulsive forces between negatively charged DMPS and PI molecules by counterions, which leads to the reduction of liposome’s size.

5.

Size distribution of particles in PBS buffer (gray), PBS containing 10^–5 mol/L CTAB (light red), and PBS containing 10^–6 mol/L CPC (light blue) measured by DLS (A) at the beginning of the experiment, (B) 120 min after the start of the experiment, (C) changes of particle size over time, and (D) changes of zeta potential of detected particles over time.

The presence of surfactants caused changes in both the particle hydrodynamic diameter and zeta potential (Figure ). Diameter values obtained for liposomes in CTAB presence (10^–5 mol/L) are 153 nm at the beginning and 123 nm at the end of the measurement, indicating an increase compared to a pure buffer environment and a slight decrease in diameter over time. As observed in pure water, CTAB molecules are suspected to accumulate close to the lipid bilayer, which results in a comparable increase in the hydrodynamic diameter. It is interesting that the observed changes for the CTAB presence are generally similar irrespective of the environment: water or PBS buffer. Quite a different situation occurs for CPCat a concentration of 10^–5 mol/L the hydrodynamic diameter was much lower than for the liposomes in pure PBS buffer (Figure S2, Table S2), indicating the possible membrane deformation, bursting, and formation of mixed micelles. Therefore, in order to keep the concentration well below the CMC of CPC in buffer (Table S1), 1 order of magnitude lower concentration (10^–6 mol/L) was used to eliminate this effect and focus on the possible interactions of the surfactant monomers with the lipid bilayer. As a result, for liposomes in such an environment, a significant increase in the hydrodynamic diameter was observed, reaching approximately 190 nm, which remained stable over time (Figure C). It suggests that CPC molecules incorporate more deeply into the lipid bilayer and in a more efficient manner than in water. The counterions present in the PBS buffer may screen the negative charges located on the phospholipid headgroups and the positive charges of the surfactants, thus preventing CPC molecules from accumulating near the headgroup layer of the outer leaflet due to electrostatic interactions and allowing them to continue the penetration process over time.

Interestingly, the zeta potential for the system with CTAB remains less negative compared to the liposomes in pure PBS, and it stabilizes at approximately −17 mV, opposite the system with water. It confirms the lower efficiency of CTAB incorporation into the outer leaflet compared to the water system, resulting from the weakening of the electrostatic lipid-surfactant interactions in the presence of counterions. In such a case, the main process would be the accumulation of CTAB molecules close to the liposomes since the energy cost of transferring CTAB into the lipid bilayer is higher compared to CPC. On the contrary, zeta potential values of liposomes in contact with CPC are comparable to or slightly lower than the zeta potential of liposomes in PBS buffer, which is consistent with the assumption of a deeper and more effective incorporation of the surfactant into the lipid bilayer.

Results obtained for both surfactants in PBS show that CPC molecules interact strongly with phospholipids, resulting in a deeper incorporation and accumulation within the lipid bilayer, while CTAB molecules accumulate close to liposomes’ surface and do not penetrate the bilayer that easily. This effect was also observed for the water environment, but the PBS buffer environment allows these differences to show more explicitly. Apparently, lipid-CTAB interactions are, in general, weaker, partly because of the bulkier structure of the CTAB headgroup. This observation is consistent with previous reports showing limited interactions of gold nanoparticles functionalized with CTAB with supported DOPC bilayers due to repulsion between CTAB and DOPC. Since this lipid is the main component of the ternary model system, it may also contribute to the limited interactions of CTAB with 3D model liposomes.

Fluorescence Microscopy of Liposomes

To complete the studies of the effect of surfactants on 3D models of the SARS-CoV-2 lipid envelope, giant unilamellar vesicles (GUVs) doped with the PC fluorescent probe were visualized using fluorescence microscopy. The changes in fluorescence intensity in the absence and presence of CTAB and CPC (10^–5 mol/L) were compared. The addition of CTAB causes a gradual increase in fluorescence, reaching a maximum after 30 min and indicating that the strongest lipid-surfactant interactions happen after a short period of time (Figure ). The shape of such vesicles is disturbed, pointing to the changes of surface tension, probably as a result of the accumulation of CTAB molecules at the outer leaflet of the lipid membrane, causing curvature tension. On the contrary, interactions in the presence of CPC can be observed immediately after the start of the measurement, demonstrating the ability of CPC to interact faster with the DOPC/DMPS/PI bilayer compared to CTAB. No disturbance of the vesicle’s shape is noted, indicating the lack of induced curvature tension in the outer leaflet. These results are consistent with the results of liposome studies and illustrate a higher potential of CPC to effectively incorporate into the 3D models of viral envelopes compared to CTAB, which tends to remain at the surface of vesicles.

6.

GUVs of the ternary model of the SARS-CoV-2 (DOPC/DMPS/PI 50:35:15) lipid envelope exposed to 10^–5 mol/L CPC and CTAB solutions and visualized by fluorescence microscopy. The scale bar is 20 μm.

Interestingly, the exposure of GUVs to surfactant solutions well above the CMC (10^–3 mol/L) did not allow us to observe a full solubilization process (Figure S3). Instead, a much brighter background was seen, and fewer liposomes could be observed in surfactant-containing systems compared to a pure PBS environment. This may suggest solubilization, as the increased background intensity from the free fluorescent probe released after liposome disruption was detected. It would be in agreement with the reduced number of GUVs, which were present in the sample, especially after 10 min in both CPC and CTAB-containing systems. These observations may be limited because of limited microscopy capability, including a limited resolution.

Conclusions

In this work, we show the physicochemical characterization of a simplified model SARS-CoV-2 lipid envelope formed both as a DOPC/DMPS/PI monolayer at the air–water interface and as a spherical liposome bilayer and the effect of two surfactants, CTAB and CPC, on such two model systems. It was shown that the electrostatic interactions of the negatively charged components, DMPS and PI, with cationic CPC and CTAB govern the interactions of surfactants with model lipid envelopes. As a result, the shift of π–A and ΔV–A isotherms of monolayers formed at the air–water interface toward larger areas per molecule was observed, indicating the penetration of the model monolayer by the surfactants. This observation is consistent with an increase in the hydrodynamic diameter and zeta potential of liposomes as well as shape fluctuations of giant unilamellar vesicles used as 3D models. However, for both model systems, the changes were shown to depend on the type of surfactant used, which allowed us to conclude that the size and structure of the headgroup of a cationic surfactant also strongly affect the interactions with model layers. CPC with the planar headgroup can penetrate the lipid layer in a more efficient way and thus influence its stability, as proved by the decreased value of surface pressure corresponding to a monolayer collapse, while CTAB with a more bulky headgroup mostly accumulates close to the layer, having problems with effective penetration. The latter observation was supported by both the changes in the shape and position of π–A and ΔV–A isotherms as well as observed fluctuations in the size and zeta potential of liposomal models. Additionally, we have shown that the presence of counterions in the PBS buffer solution and their impact on lipid-surfactant interactions and the surface activity of the surfactants themselves should be taken into consideration. The presence of counterions resulted in screening of phospholipid and surfactant charges, leading to weaker electrostatic interactions in the case of CTAB and, as a result, to comparable effects in terms of changes in the size and zeta potential of liposomes for both water and the PBS environment. It is consistent with previous literature data showing limited ability of CTAB to penetrate lipid bilayers. For CPC, a stronger effect was observed in PBS buffer, even at a lower concentration used. It is explained by the ability of CPC molecules to penetrate into the lipid mono- and bilayers, which is facilitated both by the planar structure of CPC’s polar head and by the reduction of electrostatic attractive surfactant-lipid interactions, which may prevent the deeper penetration of the surfactant into the lipid layer.

The results presented in this paper provide a more detailed mechanistic insight into the mechanisms of action of two cationic surfactants widely used in disinfecting and antiviral agents such as mouthwashes, oral rinses, and disinfecting sprays with simplified but yet accurate models of lipid envelopes of the SARS-CoV-2 virus. We have shown that even when applied at low concentrations, CPC and CTAB may change the physicochemical properties of a model lipid envelope, leading to its destabilization in specific conditions, as was also shown earlier in TEM images for CPC acting on viruses. We have also proved that the presence of counterions significantly affects the interactions of surfactants with model lipid envelopes, with the extent of these changes strongly depending on the type and molecular structure of the polar head of the cationic surfactant used. The results presented in this paper provide in-depth mechanistic insight into the mechanisms of action of cationic surfactants more from a physicochemical perspective rather than a general medical point of view, filling the gap between these two viewpoints. Therefore, this information may be useful in future work on the development of novel, effective antiviral and disinfecting agents, since the viral lipid envelopes are responsible for the protection of the genetic material of the virus inside the capsid, and therefore, their destabilization or changes in physicochemical properties may finally lead to the destruction of the virus, resulting in the reduced infectivity.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.langmuir.6c00207.

• Hydrophilic–lipophilic balance (HLB) and critical micelle concentration (CMC) values of CTAB and CPC surfactants in aqueous solutions; surface pressure–area per molecule (π–A) and surface potential–area per molecule (ΔV–A) isotherms of surfactant monolayers on PBS buffer subphase; changes of hydrodynamic diameter and zeta potential in time of DOPC/DMPS/PI 50:35:15 liposomes in PBS and 10^–5 M CPC measured by DLS; characteristic parameters of DOPC/DMPS/PI 50:35:15 liposomes in PBS containing 10^–5 M CPC measured by DLS; and fluorescence microscopy images for GUVs exposed to surfactant solutions above CMC (PDF)

The authors declare no competing financial interest.

Data are available in the data repository https://doi.org/10.58132/1EJXFW.