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Article

Experimental Study of the Interaction of Silica Nanoparticles with a Phospholipid Membrane

by
Viktoria Milkova
Rostislaw Kaischew Institute of Physical Chemistry, Bulgarian Academy of Sciences, ‘Acad. G. Bonchev’ Str., Bl. 11, 1113 Sofia, Bulgaria
Sci 2025, 7(1), 6; https://doi.org/10.3390/sci7010006
Submission received: 21 November 2024 / Revised: 21 December 2024 / Accepted: 31 December 2024 / Published: 3 January 2025
(This article belongs to the Section Chemistry Science)
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Abstract

:
This study aims to contribute to the physical understanding of the behavior of nanoparticles in lipid–nanoparticle composite systems. Therefore, composite films were formed on hydrophilic or hydrophobic surfaces through the sequential adsorption of liposomes and silica nanoparticles. The process was performed using dispersions with different pHs by using optical fixed-angle reflectometry. In the first step, liposomes were deposited on the surface, resulting in a lipid monolayer or bilayer depending on the surface’s properties. The kinetic experiments indicated that the adsorption of liposomes is a diffusion-limited process that depends on the pH and the properties of the substrate. In the second step, negatively charged nanoparticles were adsorbed on the membrane as a result of the electrostatic interactions with the positively charged domains on the membrane. The amounts of liposomes and particles adsorbed depend on the charge density of the particles and net charge density of the membrane: an increase in the pH and hydrophobicity of the surface leads to a decrease in the amounts adsorbed because of the increase in the electrostatic repulsion between particles and lipids. The procedure was supplemented with the formation of two liposomes/nanoparticles bilayers.

1. Introduction

Nanoparticles (NPs) are the bridge between macroscopic and atomic or molecular structures. The surface-to-volume ratio plays a crucial role in their physicochemical, optical and biological properties [1,2]. At the same time, with the increasing widespread application of NPs in pharmaceuticals, medicine, cosmetics, food products, packaging and electronics, as well as the difficulty of controlling their disposal, the need for environmental and health risk studies has increased. Other sources of NPs for the human body are medical implants and contrast agents used in some clinical imaging protocols. NPs may enter the blood stream by permeating through the skin [3,4]. There is a contradiction in the information about the toxicity of NPs, but recently, some studies have shown that there is a relationship between the size of NPs and their cytotoxicity [5]. Therefore, the interaction of phospholipid membranes (as a model of cell membranes) with nanomaterials (nanostructured materials and nanoparticles) is relevant from a pharmaceutical and toxicological viewpoint [6,7,8,9,10]. On the other hand, this knowledge is important for understanding the transport of NPs through the membrane because of their application as delivery systems.
Silica nanoparticles are popular model particles that can be synthesized due to their appropriate size and very narrow size distribution. These particles have been extensively studied as stabilizers of emulsion droplets or liposomes. The unique characteristics of silica nanoparticles (optical properties, high specific surface area, low density, adsorption capacity for encapsulation, biocompatibility and low toxicity, highly controllable size and shape) make them suitable for various applications in biotechnology, but also for a wide range of model investigations. Moreover, “liposils” (developed from liposomes and silica) are structures with promising potential as delivery systems for vitamins or bio-active components [11,12,13].
Very small silica NPs (up to 20 nm) can be used as a model of protein molecules to simulate their interaction with a model membrane or used for the development of structures like a protein corona. The price of commercial pure proteins is extremely high for a few micrograms, which hinders the implementation of systematical protein studies that have a methodology and procedure requiring a high concentration or volume of protein solutions. The analysis of the effect of solid NPs on the properties of the model membrane is the object of extensive experimental and theoretical studies that present different aspects of the process: the effect of the size and charge of the particles, the hydrophilicity or hydrophobicity of NPs, the modification of the particle surface (polymer adsorption), the influence of the charge and composition of the membrane, the elasticity or thickness of the hydrophobic part of the lipid bilayer, etc. [6,7,8,9,10].
Supported lipid bilayers (SLBs) have been used in many studies concerning cell–cell interactions or the adsorption of proteins as a model of biological membranes. Generally, SLBs can be formed by liposome deposition on a solid substrate. Despite the considerable studies on liposome adsorption, we lack a complete picture of the forces responsible for the adhesion, fusion and rupture of liposomes on solid surfaces. The formation is governed predominantly by three types of interactions: (i) the interaction between the liposomes and substrate; (ii) the molecular interactions in the bilayer of the adsorbed liposomes characterized by the bending modulus; and (iii) the interaction between the adsorbed liposomes (hydrophobic, steric and electrostatic forces). Liposomal rupture and the formation of a full SLB will occur if the liposome deformation exceeds a given threshold value and if critical coverage is achieved on the substrate [14].
Lipowsky et al. [15] proposed a model for SLB formation using unilamellar liposomes. The process starts with the adhesion of liposomes on the surface, governed by the effective contact potential. Subsequently, the adhesion induces topological changes in the liposomal morphology, leading to liposome rupture and fusion. According to the model, the liposomes can undergo transformation between the different states if their radius is larger than the critical radii defined for these transitions. The charge of lipids affects the balance between the interactions on the surface and is a determining factor for the vesicle deposition pathway.
Many theoretical and experimental studies have shown that liposome adsorption is irreversible and often controlled by diffusion [15], and that the formation, stability and properties of SLBs strongly depend on many parameters, such as the substrate type and surface charge density, surface roughness, pH, ionic strength, presence of the divalent cations, temperature, lipid concentration, lipid composition, charge of the lipid head group and liposomal size [16,17,18,19,20].
Several methods for the formation of SLBs onto solid surfaces have been described in the literature; these include Langmuir–Blodgett deposition [21], spin coating, and adsorption from mixed micelles [22]. The formation of supported lipid bilayers by vesicle fusion on hydrophilic surfaces was introduced by McConnell at al. [23]. This approach is attractive because of its simplicity, reproducibility and almost defect-free surface coverage of the lipid layer. The formation of SLBs on silica nanoparticles was reported for the first time by Mornet et al. [24].
The present study investigated the interaction of well-defined model silica NPs and a planar zwitterionic membrane by using reflectometry. The nanoparticles are used as a model for bioactive components, with a well-defined and permanent structure suitable for entrapment into a soft membrane-like platform. The effect of the particle charge was studied via an analysis of the mechanism of the formation of a multilayer lipid/particles system on hydrophilic and hydrophobic solid surfaces. The results obtained contribute to our understanding of the behavior of silica NPs in lipid–nanoparticle composite systems.

2. Materials and Methods

2.1. Formation of the Liposomes

The liposomes were produced from the phospholipid 1,2-dioleoyl-sn-glicero-3-phoshocholine (DOPC, chloroform solution, 25 mg/mL, Avanti Polar Lipids Inc., provided by Sigma-Aldrich, Taufkirchen, Germany) by using the thin film hydration method. The procedure was described in a previous study [25]. Very briefly, an appropriate volume of the lipid solution (200 μL) was dried under a stream of nitrogen by rotating the flask to form a thin lipid film on its wall. The lipid was re-hydrated in a buffer solution (2 mL, 10 mM buffer pH 4, 7 or 9) to a final lipid concentration of 2.5 mg/mL. After subjecting the tube to 4 freeze–thaw cycles via dispersion in a bath of liquid nitrogen and hot tap water, the stock solution of liposomes was sonicated in an ultrasonic ice bath for 15 min.
To prevent possible aggregation during the experimental procedure, the dispersion was prepared by mixing 80 µL of the stock dispersion of liposomes and 10 mL of the buffer solution to obtain a final lipid concentration of 0.02 mg/mL. The dispersion was filtered before measurements were performed by extrusion through a 0.4 µm filter (Minisart®, Sartorius, Gottingen, Germany). The liposome suspensions were stored at −20 °C and were remarkably stable for at least three months.
The liposome dispersion used for the reflectometry experiments was prepared by mixing 1 mL of the stock dispersion of liposomes and 249 mL of buffer with a suitable pH (4, 7 or 9) to obtain a final lipid concentration of 0.01 mg/mL. The dispersion was filtered 4 times before measurements were performed by extrusion through a 0.22 µm filter (Minisart®, Sartorius).

2.2. Nanoparticles

The suspensions of silica particles Ludox LS30 and Ludox TM50 (0.03 wt%) were prepared in plastic flasks containing the stock suspension, with buffer with a pH of 4, 7 or 9 topping up the flask to 250 mL. Silica particles were purchased from Sigma–Aldrich, Taufkirchen, Germany.

2.3. Preparation of Silicon Wafers for Formation of the Planar Bilayers

Silicon wafers were baked for 90 min at 1000 °C to form a silica layer of about 100 nm (checked by ellipsometry) on their surface, which is essential for obtaining high sensitivity in reflectometry experiments. The Si/SiO2 wafers were cut into strips (1 × 5 cm). To investigate deposition on hydrophilic SiO2 surfaces, the strips were cleaned in a plasma cleaner and thoroughly rinsed with ethanol and deionized water before each experiment.
Strips of the Si/SiO2 wafers were made hydrophobic via treatment with hexamethyldisilazane (HMDS) after being cleaned with water, ethanol and a plasma cleaner, respectively. The strips were then placed in a desiccator, with 2 mL of HDMS in a small petri dish at the bottom of the desiccator. After removing the air from the desiccator by streaming nitrogen gas, the desiccator was closed and the strips were left to react with the HDMS for at least 12 h until use. Just before the reflectometer measurements, the strips were taken out, washed with chloroform and ethanol to remove any excess HDMS, and dried with nitrogen gas. In water, the hydrophobic surface remains completely stable up to approximately 200 min (water contact angle > 90°) [26].

2.4. Reflectometry

Liposome deposition on silicon wafers for the formation of supported lipid layers and the sequential adsorption of nanoparticles was performed by using an optical fixed-angle reflectometer with a stagnation point flow cell, as described by Dijt et al. [27,28]. The source of monochromatic light (He-Ne laser, λ = 632.8 nm) was linearly polarized and passed through a 45° glass prism. The beam propagated to the substrate surface at an angle of incidence of 70°, close to the Brewster angle θb = arctan (n2/n1), where the n1 and n2 are the refractive indexes of the solvent and substrate, respectively.
In this setup, the solution flows from a tube perpendicular to a strip of oxidized silicon wafer. The laser beam reflects from the surface at exactly the point where the jet flow reaches the surface. The limiting flux J of the adsorbing species in this stagnation point can be calculated using the following [29]:
J = 0.283 ν − 1 / 3 Φ 2 / 3 R − 5 / 3 D t 2 / 3 c
where ν is the kinematic viscosity of the solution/dispersion, Φ is the volume flux, R is the radius of the tube of the impinging jet system (for our setup this is 9.5 × 10−4 m), Dt is the translation diffusion coefficient of the adsorbing species, and c is its concentration in bulk solution. The solutions/suspensions used here are very dilute, which allows ν to be used as the value for pure water: 10−6 m2/s. The volume flux Φ to the surface was in the order of a few mL/min.
Upon adsorption, the reflectometer signal changes, and the procedure used to calculate the amount adsorbed Γ from this signal change has been described by Dijt et al. [27,28]. For the refractive index increment dn/dc of the adsorbing vesicles and silica nanoparticles, we used values of 0.146 mL/g [30] and 0.061 mL/g (measured using an Abbe refractometer), respectively. In the case of transport (diffusion)-limited adsorption, in the initial stage of the adsorption process, all vesicles or particles that arrive at the surface adsorb, so that the limiting flux J is equal to the initial adsorption rate (dΓ/dt)t = 0.
The analysis of the reflectometry results was combined with the ellipsometry measurements. The thickness of the oxide layer on the wafers and the thickness of the coverage after each deposition step were evaluated.

2.5. Formation of the Sandwich-like Film from Lipids and NPs on the Membrane

The film was formed in the reflectometry cell on the suitable silicon wafers by using dispersions and buffer solutions with a pH of 4, 7 or 9. The first step in film formation is injection into the liposome solution (10−2 mg/mL). The duration of the injection was enough to ensure that the deposited amount was saturated. Then, the surface was rinsed with buffer with the same pH for a few minutes. The next deposition step was performed by injecting the system with the nanoparticle dispersion (0.03 wt%). The next deposition layers were formed using the same method.

2.6. Determination of the Electrokinetic Charge and Size of Liposomes and NPs

The properties of the produced liposomes and NPs were assessed using dynamic light scattering with non-invasive backscattering (DLS-NIBS) at a measuring angle of 173°. The Zetasizer Pro (Malvern, UK), equipped with a He-Ne laser with a maximum power of 10 mW and operating at a wavelength of 633 nm, was used to carry out the measurements. Each sample was measured five times, and the average value was recorded as the final size and surface charge.

2.7. Microscopy

The morphology of the lipid bilayer (a) and bilayer in the presence of NPs (b) was studied with a field emission scanning electron microscope (JEOL IT800SHL, JEOL Ltd., Tokyo, Japan), using both secondary and backscattering electron detectors placed within the in-chamber of the lens microscope columns.

3. Results

3.1. Characterization of the Liposomes and Particles

The hydrodynamic size (diameter) and charge of the liposomes and NPs at different pHs are presented in Table 1.

3.2. A Sequential Adsorption of Liposomes and NPs

Figure 1 presents the sequential deposition of liposomes and silica nanoparticles on the hydrophilic and hydrophobic surfaces.
The first plateau region in each curve indicates the achievement of the maximum absorbed amount of lipids on the surface. According to the presented results, the plateau values of the adsorption on the hydrophilic surface (ca. 3.5 mg/m2) do not depend on the pH of the solution. However, saturated adsorption is achieved in 5 min (at pH 4), 9 min (at pH 7) and 100 min (pH 9) after the injection of the liposomes. The summarized results are presented in Table 2, Table 3 and Table 4.
The amount deposited on the hydrophobic surface is about twice as low, which points to the formation of a single monolayer (ca. 1.7 mg/m2 at pH 4 and pH 7, ca. 0.7 mg/m2 at pH 9). The rinsing of the surface through the injection of a buffer solution (with the same pH) in the system (ca. 5–10 min) did not lead to the significant desorption of lipids.
The second plateau in each curve presented in Figure 2 is achieved when the adsorption of NPs is saturated on the membrane. The results indicate a sharp increase in the number of particles on the surface. The maximum absorbed amount depends on the pH of the dispersion and is achieved at approximately 35 min (at pH 4), 45 min (at pH 7) and 137 min (at pH 9) after the injection of the nanoparticles in the system.
Figure 2 and Figure 3 present the reflectometry results obtained for the composite film formed through the sequential adsorption of liposomes and silica NPs on the hydrophilic or hydrophobic surface at pH 4 and pH 7, respectively. In total, four layers are deposited, starting with the adsorption of lipids. The plateaus in the reflectometry curves indicate that, for each adsorption step, the maximum adsorbed amount is achieved. The first and second plateau regions correspond to the formation of a lipid mono- or bilayer and the sequential adsorption of NPs, respectively, as described above. The achievement of saturated adsorption in the formation of the second lipid layer (in the third step) requires approximately 30 min, which is significantly longer than the time required for the adsorption of the first layer (up to 10 min for pH 4 and pH 7) (Table 2, Table 3 and Table 4).

4. Discussion

The reflectometry experiments give important information about the kinetics of the adsorption of the liposomes and NPs. From the initial slopes of the curves presented in Figure 1, the increment in the amount adsorbed from lipids can be estimated (Table 1 ca. 1 mg/m2 min for the hydrophilic and hydrophobic surfaces). These values are in the same order as the limiting flux estimated using Equation (1). In the calculation, the values of ν (10−6 m2/s), Φ (0.067 mL/s), R (9.5 × 10−4 m), c (0.01 mg/mL) and the translation diffusion coefficient of liposomes from their radius (r = 100 nm) are estimated by using the Stokes–Einstein equation ( D t = k B T 6 π η r ).
Therefore, it can be concluded that, for the medium with these pH values, the formation of the lipid layers is limited by diffusion. At pH 9, the initial increment in the adsorption rate is about 200 lower, indicating an energy barrier of about 5 kT for the adsorption of liposomes on the hydrophilic silica surface.
It is well known that the charge of the silica depends on the pH (ZPC is 2.9 [31]) and that, with the increase in the pH of the solution, the negative charge of the surface also increases. Simultaneously, the net charge of the membrane (formed from DOPC, which is a zwitterion lipid) at a high pH is also negative [32]. Therefore, the electrostatic hindrance of the lipid adsorption on the similarly charged surface is expected.
The results in Figure 1 show that the maximum amount of lipids adsorbed on the hydrophilic silica surface (DOPC 1) is about 3.5 mg/m2, corresponding to approximately 7.0 × 1018 lipid molecules in the bilayer, which is in line with other studies [33,34,35]. Using a head group area of 0.8 nm2 for phosphatidylcholine [36], the full coverage of the surface by a single bilayer corresponds to ~9.7 mg/m2. Therefore, it can be concluded that a full lipid bilayer is formed on the used surface.
For comparison, the amount of lipids deposited on the hydrophobic silica surface (open symbols in Figure 1) is about two times lower, which points to the formation of a single monolayer. In addition, the initial adsorption rates are considerably lower than in the case of the untreated hydrophilic silica surface (Table 1). It is clear that, on the hydrophobic surface, the lipid deposition rate is not transport limited, but determined by the attachment and rupture rate of the liposomes. This can be understood based on a model of lipid monolayer formation on a hydrophobic surface by liposome fusion proposed by Kalb et al. [37]. According to this model, when a liposome approaches the surface, a defect in the outer lipid monolayer occurs, leading to hydrophobic contact between the hydrophobic part of the bilayer and the surface. Subsequently, the outer monolayer spreads and leads to the rupture of the liposome on the surface. Monolayer formation via DOPC vesicle deposition on hydrophobic silica has also been reported by Santos et al. [29]. In the next adsorption step presented in the absorption curves in Figure 1, NPs are adsorbed on the lipid membrane due to the attractive interactions between zwitterionic liposomes and silica NPs.
It was found that the particles were immersed or deposited on the top of the lipid layer and that the hydrophilicity of the substrate was relevant for this adsorption step. The lipid bilayer formed on the hydrophilic surface was probably more dynamic compared to the monolayer on the hydrophobic surface. It was found that a thin layer of water and ions were present between the lipid bilayer and the surface, to compensate for the surface charge of the substrate, while the tails of the lipid molecules forming the monolayer were in close contact with the substrate by hydrophobic binding.
The number of particles adsorbed from NPs on both substrates gradually decreased as the pH of the dispersion increased (Table 1, Table 2 and Table 3). According to the presented results, the number of particles adsorbed on hydrophilic surfaces is ca. 17.8 mg/m2 (at pH 4), ca. 10.8 mg/m2 (at pH 7) and ca. 4.9 mg/m2 (at pH 9). This is mainly due to the increasing negative surface charge density of the silica nanoparticles with pH, which varies from a few mC/m2 at pH 4 to about −300 mC m−2 at pH 9 [38].
The negative charge of the particles increases with the pH of the solution. It was supposed that the interaction between the particles and membrane resulted from the electrostatic attraction between the negatively charged NPs and positively charged domains (groups) on the lipid membrane.
The simple estimation showed that full coverage of the surface can be achieved with ca. 18.4 mg/mL silica NPs (in the calculation, the density of the silica was 2.36 mg/m3 and the weight of one silica nanoparticle was 5 × 10−15 mg). The comparison with the experimental data indicates that almost full coverage on the first lipid layer is achieved at pH 4 (ca. 96%) and that very low coverage is registered at pH 9 (ca. 27%).
The experimental results for the thickness of the formed composite film indicate that the thickness of the lipid bilayer (ca. 7.3 nm) and monolayer (ca. 3 nm) does not depend on the pH of the dispersion, but that the thickness depends only on the surface properties. However, the thickness of the layer of nanoparticles slightly depends on the pH. The greater number of NPs adsorbed at pH 4 correlates (Figure 1) with the increase in the thickness. It was supposed that there is a relation between the thickness and the almost full coverage of the lipid layer with NPs at pH 4 (ca. 96%). Some of the particles are loosely deposited on the surface. Some of these particles will be removed during the rinsing step, but some of them will be present in the layer. At pH 7 and pH 9, respectively, a low coverage of NPs (ca. 27%) is registered, and there is enough surface for all the adsorbed particles on the lipid layer. Moreover, information about the thickness, adsorbed amount and coverage of the lipids and NPs indicates that in the present experimental conditions, only one bilayer or one monolayer and one layer from particles is formed at each deposition step.
Remarkably, the amount of lipids adsorbed (~10 mg m−2) in the third adsorption step (DOPC 2) is almost equal to the estimated amount of lipids when there is full coverage of the surface (for bilayer is ~9.69 mg m−2). It should be noted that without nanoparticles, the deposition of a second lipid bilayer would be completely suppressed, as shown by Oleson et al. [39]; this is because the electrostatic and van der Waals interactions are too weak beyond the first bilayer. It can be assumed that the higher amount of lipids adsorbed in the third step is due to a relatively large adsorbing surface area, as a result of the presence of NPs (surface roughness), and the deformation of (folds in) the bilayer that is deposited in this step.
The analysis of the results presented in Figure 2 and Figure 3 shows that the increase in the amount of lipids adsorbed in the third deposition step on the hydrophilic surface is approximately equal to the limiting flux of the liposomes. On the hydrophobic surface, these rates are still significantly lower and the time required to achieve the plateau values in the adsorbed amounts is about twice as high as that for the hydrophilic substrate. At the same time, the adsorbed amounts are almost the same. It was supposed that the decrease in the adsorption rate is probably related to rearrangements in the adsorbed layers, which are easier and faster at the hydrophilic surface than at the hydrophobic surface.
The question is whether the second bilayer envelops the nanoparticles or whether the particles penetrate the bilayer. The wrapping of the bilayer around nanoparticles is determined by a balance between the (favorable) adhesion energy between the membrane and the particle and the (unfavorable) elastic energy required to bend the bilayer around the particle [14]. Various theoretical studies have provided phase diagrams showing the regions of fully enveloped, partially wrapped or freely dispersed particles [40,41]. According to the model of Helfrich et al. [42], this can be realized if the radius of the particle, RC, is higher than a critical value. The transition between the wrapped and the free state of particles is characterized by a critical radius for the particle, meaning that the particle wrapping is energetically favorable for particles with a radius above this critical value. The experimentally determined values for this critical radius have been found to vary with the nature of the system (type of lipids and particle surface, pH and ionic strength).
Experimentally, Roiter et al. [43] studied the adsorption of lipid vesicles on nanostructured surfaces prepared by decorating silicon wafers with silica particles of different sizes. From the analysis of AFM images and using data from simulation studies on the interaction of colloids with lipid membranes, the authors reported different adsorption behaviors above and below the critical particle diameter, RC (ca. 10–15 nm). The experimental studies have confirmed that particles with diameter D >> RC are mostly covered by the deposited lipid bilayer, but if D << RC, the particles seem to ‘pierce’ the bilayer.
Thus, the LS30 NPs used in the present study have a diameter of about 16 nm (based on data from the manufacturer and from the DLS measurements performed), so one can expect them to penetrate through the lipid layer.
The distinction of small individual particles from the images obtained from SEM (Figure 4) is very speculative. It was supposed that the bigger visible particles on the membrane are aggregates (ca. 100 nm), which are formed during the drying of the samples for microscopy.
The fourth plateau regions in Figure 2 and Figure 3 correspond to the adsorption of nanoparticles (NPs 2). The estimated amounts are compared in Table 1 and Table 2. For the hydrophilic and hydrophobic surfaces, the adsorbed amounts do not depend on the pH of the suspension, but the adsorbed amounts on the hydrophobic surface are considerably lower, at only ~5 mg m−2, corresponding to 1 × 1015 particles per m2. This is surprising since, in the second step, the nanoparticles did not seem to be affected by differences between the bilayer formed on the hydrophilic or hydrophobic silica surface. The dynamics of rearrangements in the multilayer structure become more important after the deposition of an extra lipid bilayer.
Using larger silica particles (radius ~31 nm) leads to a higher adsorption of particles, not only in mass but also in number density (~33 mg m−2 corresponding to 1.1 × 1014 m−2 and 55 mg m−2, corresponding to 1.9 × 1014 m−2, respectively). This is probably because larger particles have a less disturbing effect on the lipid bilayers since their curvature is reduced and they have a lower tendency to penetrate the bilayers (Figure A1).

5. Conclusions

The interaction between negatively charged silica NPs and zwitterionic DOPC mono- and bilayers are studied through the formation of lipid/nanoparticle films on hydrophilic or hydrophobic surfaces stabilized predominantly by electrostatic interactions.
At a low pH, the deposition rate of the liposomes and formation of a bilayer on the hydrophilic surface is approximately equal to the limiting flux, indicating that the process is transport limited. At a higher pH, the rate is considerably lower due to the increase in the negative charge density of the silica surface. The liposome adsorption rate on the hydrophobic surface is significantly lower than the limiting flux, probably because a defect in the outer bilayer must occur before there is hydrophobic contact between the liposome and the surface.
The deposition of NPs on the lipid membrane is due to the attractive electrostatic interactions between zwitterionic liposomes and silica, and does not depend on the properties of the substrate. The number of NPs adsorbed on hydrophobic or hydrophilic substrates gradually decreases as the pH of the dispersion increases because of the increase in the electrostatic repulsion between the negatively charged lipids and silica particles. The higher number of NPs adsorbed at a low pH correlates with the increase in the thickness. Moreover, the experimental results about the thickness, adsorbed amount and coverage of the lipids and NPs indicate that in the present experimental conditions, only one bilayer, one monolayer and one layer is formed from particles at each deposition step.
The experimental results obtained indicate that the electrical properties (surface charge density) of the lipids and nanoparticles define the interaction between them. At a low pH, the interactions can be explained through weak electrostatic interactions and the formation of hydrogen bonds between lipids with a very low positive charge density and silica particles with a very low negative surface charge density, whereas at a high pH, the electrostatic repulsion between sternly negatively charged particles and lipids dominates.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Acknowledgments

The reflectometry experiments were performed within the framework of the EU project ENNSATOX (Project 229244) funded by the European Commission in Wageningen University & Research, Physical Chemistry and Soft Matter, Wageninegen, The Netherlands.

Conflicts of Interest

The author declare no conflicts of interest.

Appendix A

Figure A1 presents the formation of a lipid/NPs composite structure on the hydrophilic surface by using larger silica particles (Ludox TM50, radius 31 nm) at pH 4.
Sci 07 00006 g0a1
Figure A1. Sequential adsorption of DOPC liposomes and TM50 silica NPs on hydrophilic surface at pH 4.
Figure A1. Sequential adsorption of DOPC liposomes and TM50 silica NPs on hydrophilic surface at pH 4.
Sci 07 00006 g0a1

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Figure 1. Sequential deposition of liposomes and silica LS30 nanoparticles on a (a) hydrophilic and (b) hydrophobic surface. Dependence of the adsorbed amount when using 10 mM of buffer at pH 4 (●○), pH 7 (●○) and pH 9 (●○). (c) The estimated number of adsorbed lipid molecules (■□) and nanoparticles (●○) expressed in species/ m2 on the surface, as a function of the pH of the dispersion. (d) The estimated thickness of the lipid bilayer and layer of nanoparticles formed on the hydrophilic and hydrophobic (inset) surfaces. In the calculations, the weight of one silica nanoparticle is 5 × 10−15 mg and the molecular weight of DOPC is 786.1 g/mol.
Figure 1. Sequential deposition of liposomes and silica LS30 nanoparticles on a (a) hydrophilic and (b) hydrophobic surface. Dependence of the adsorbed amount when using 10 mM of buffer at pH 4 (●○), pH 7 (●○) and pH 9 (●○). (c) The estimated number of adsorbed lipid molecules (■□) and nanoparticles (●○) expressed in species/ m2 on the surface, as a function of the pH of the dispersion. (d) The estimated thickness of the lipid bilayer and layer of nanoparticles formed on the hydrophilic and hydrophobic (inset) surfaces. In the calculations, the weight of one silica nanoparticle is 5 × 10−15 mg and the molecular weight of DOPC is 786.1 g/mol.
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Figure 2. Sequential adsorption of DOPC liposomes and LS30 silica NPs on hydrophilic (a) and hydrophobic (b) surfaces at pH 4.
Figure 2. Sequential adsorption of DOPC liposomes and LS30 silica NPs on hydrophilic (a) and hydrophobic (b) surfaces at pH 4.
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Figure 3. Sequential adsorption of DOPC liposomes and LS30 silica NPs on hydrophilic (a) and hydrophobic (b) surfaces at pH 7.
Figure 3. Sequential adsorption of DOPC liposomes and LS30 silica NPs on hydrophilic (a) and hydrophobic (b) surfaces at pH 7.
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Figure 4. Visualization of the deposited lipid bilayer without silica particles (a) and bilayer with 30 LS NPs (b) on a hydrophilic surface, pH 4 (the bars correspond to 100 nm). Inset: The scale bar on the image corresponds to 50 nm.
Figure 4. Visualization of the deposited lipid bilayer without silica particles (a) and bilayer with 30 LS NPs (b) on a hydrophilic surface, pH 4 (the bars correspond to 100 nm). Inset: The scale bar on the image corresponds to 50 nm.
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Table 1. Characteristics of the silica NPs LS30 and produced liposomes for dispersions with different pHs.
Table 1. Characteristics of the silica NPs LS30 and produced liposomes for dispersions with different pHs.
SampleD, nm
(PDI)		ζ-Potential
pH 4pH 7pH 9pH 4pH 7pH 9
liposomes223.7 ± 3.9
(0.12)		198.4 ± 7.4
(0.22)		200.5 ± 2.6
(0.10)		−22.1 ± 0.4−70.5 ± 1.2−83.2 ± 2.7
NPs19.3 ± 1.2
(0.14)		20.4 ± 2.0
(0.10)		18.1 ± 1.7
(0.31)		−26.2 ± 1.0−95.0 ± 4.5−135.1 ± 1.9
Table 2. Summarized results from the reflectometry experiments for the sequential adsorption of DOPC liposomes and LS30 silica NPs at pH 4.
Table 2. Summarized results from the reflectometry experiments for the sequential adsorption of DOPC liposomes and LS30 silica NPs at pH 4.
SurfaceAdsorption StepΓexp saturated
mg/m2
Hydrophilic
pH 4		DOPC 13.5
NPs 117.8
DOPC 29.1
NPs 225.1
Hydrophobic
pH 4		DOPC 11.6
NPs 115.9
DOPC 210.8
NPs 25.9
Γfull coverage (lipids for bilayer)~9.7 mg/m2, Γfull coverage (NPs LS30)~18.4 mg/m2.
Table 3. Summarized results from the reflectometry experiments for the sequential adsorption of DOPC liposomes and LS30 silica NPs at pH 7.
Table 3. Summarized results from the reflectometry experiments for the sequential adsorption of DOPC liposomes and LS30 silica NPs at pH 7.
SurfaceAdsorption StepΓexp saturated
mg/m2
Hydrophilic
pH 7		DOPC 13.5
NPs 110.8
DOPC 29.1
NPs 220.7
Hydrophobic
pH 7		DOPC 11.7
NPs 111.2
DOPC 27.1
NPs 24.6
Γfull coverage (lipids for bilayer)~9.7 mg/m2, Γfull coverage (NPs LS30)~18.4 mg/m2.
Table 4. Summarized results from the reflectometry experiments for the sequential adsorption of DOPC liposomes and LS30 silica NPs at pH 9.
Table 4. Summarized results from the reflectometry experiments for the sequential adsorption of DOPC liposomes and LS30 silica NPs at pH 9.
SurfaceAdsorption StepΓexp saturated
mg/m2
Hydrophilic
pH 9		DOPC 13.5
NPs 14.9
Hydrophobic
pH 9		DOPC 10.7
NPs 17.5
Γfull coverage (lipids in monolayer)~4.8 mg/m2, Γfull coverage (NPs LS30)~18.4 mg/m2.
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Milkova, V. Experimental Study of the Interaction of Silica Nanoparticles with a Phospholipid Membrane. Sci 2025, 7, 6. https://doi.org/10.3390/sci7010006

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Milkova, V. (2025). Experimental Study of the Interaction of Silica Nanoparticles with a Phospholipid Membrane. Sci, 7(1), 6. https://doi.org/10.3390/sci7010006

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