Lipid-based mucus penetrating nanoparticles and their biophysical interactions with pulmonary mucus layer

European Journal of Pharmaceutics and Biopharmaceutics 149 (2020) 45–57

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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Lipid-based mucus penetrating nanoparticles and their biophysical interactions with pulmonary mucus layer Gokce Alpa, Nihal Aydoganb, a b

T

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Department of Chemical Engineering, Hacettepe University, Beytepe 06800, Ankara, Turkey Department of Chemical Engineering, Hacettepe University, Beytepe 06800, Ankara, Turkey

A R T I C LE I N FO

A B S T R A C T

Keywords: Pulmonary drug delivery Solid lipid nanoparticles Mucus Mucus-particle interactions Penetration

Lungs are critical organs that are continuously exposed to exogeneous matter. The presence of the mucus layer helps to protect them via its adhesive structure and filtering mechanisms. Mucus also acts as a strong barrier against the drugs and nanocarriers in drug delivery. In this study, solid lipid nanoparticles (SLNs), at different sizes and surface properties, were prepared and their spreading/penetration ability was tested for their use in pulmonary drug delivery. The biophysical interactions of SLNs have been studied via light scattering (LS) and zeta potential analyses by incubating the SLNs in mucin solution and forming a model mucus layer using a Langmuir-Blodgett (LB) trough. In addition, the penetration performance of the particles was evaluated using Franz diffusion cell and rotating diffusion tubes. It was determined that 36% of SLNs can penetrate through a 1.2 ± 0.2-mm-thick mucus layer. Finally, the spreading behavior of the particles on a mucus-mimicking subphase was characterized and enhanced using a catanionic surfactant mixture. Overall, the current study was the first to investigates both the spreading and penetration performance of SLNs. The developed systems offer a drug delivery system that is able to achieve high penetration rates through a thick mucus layer.

1. Introduction The pulmonary route is considered an advantageous route for drug administration owing to the very large surface area and absorption characteristics of the lungs [1–3]. However, the upper airways are covered with a mucus layer, which serves as a barrier to exogenous matter and provides a natural defense for the immune system as a result of its self-cleaning mechanism (mucociliary clearance). Therefore, it is difficult to achieve a successfully functioning drug delivery system through the lungs [4–6]. It is known that pulmonary mucus in healthy individuals contains 95% water, 0.5–5% glycoproteins and lipids, 0.5%1% mineral salts, and approximately 1% protein [7]. Although the thickness of this layer in pulmonary airways varies, it has been reported that the average thickness of the mucus is in the range of 10–30 μm at the trachea and 2–5 μm at the bronchus [8]. During the course of several lung diseases such as chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF), the thickness and the composition of the mucus layer increases, and this change in the biophysical parameters of the barrier becomes a real challenge towards the diffusion of drug carriers and the delivery of therapeutics [9–12]. In this case, traditional aerosolized drugs or drug carriers can be ineffective and a more efficient way to provide a better treatment should be considered [13,14].

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To obtain an effectively functioning drug delivery system, transporting a sufficient amount of drug to the targeted region is vital. In order to achieve better diffusion of drug carriers, primarily interactions between the drug carriers and mucus should be analyzed and understood well. After drug carrier particles reach the mucus layer, a corona is formed on the surface of the particles, depending on the surface properties of the particles. Here, in particular, mucoglycoproteins-mucins are the key macromolecules responsible for the structural properties of mucus and they determine its function as a molecular sieve [15,16]. It is known that the penetration of drugs, particles, or nanocarriers is considerably hindered by the mucus through several interactions between the particles and the mucus layer [17]. Specifically, 2 types of filtering mechanisms are employed by the mucus layer towards drug delivery systems, which are categorized as size and interaction filtering mechanisms. As a result of electrostatic and hydrophobic attractive forces, mucus can adsorb on particles or drug carriers, which is called the interaction filtering mechanism. Noninteracting nanoparticles (NPs) that are smaller in size than the pore size of the mucus can penetrate through the pores, in contrast to the larger-sized particles that are immobilized in the network (size filtering) [18–20]. Studies have reported that the overall particle mobility depends mainly on the mucus composition, particle size, particle coating,

Corresponding author. E-mail address: [email protected] (N. Aydogan).

https://doi.org/10.1016/j.ejpb.2020.01.017 Received 29 May 2019; Received in revised form 25 October 2019; Accepted 29 January 2020 Available online 31 January 2020 0939-6411/ © 2020 Elsevier B.V. All rights reserved.

European Journal of Pharmaceutics and Biopharmaceutics 149 (2020) 45–57

G. Alp and N. Aydogan

investigated in detail via a Langmuir trough, by changing the surface pressure of the interface. This provided the mucus layers with different densities and thicknesses, so as to obtain preliminary results for the penetration abilities of the particles, which had been implemented for the first time herein. This method had provided us a straightforward result about the penetration and diffusion behaviors of particles through the mucus layer for different layer thicknesses. The resulting interfaces were transferred to clean microscope slides and evaluated via atomic force microscopy (AFM) to visualize the form of the mucin layer and the presence of the particles at the interface. Penetration of the particles through the mucus layer was also studied in 3-dimensionally (3D) via the Franz diffusion cell and rotating diffusion tube methods. Finally, to model a complete drug delivery system, the spreading behavior of the particles on a mucus-mimicking surface was studied, and even enhanced for the first time herein using a catanionic formulation that was recently developed [40]. One of the important points that should be emphasized is that these newly formulated SLNs had been used as-synthesized, without any further modifications. Therefore, an additional advantage of this study was the simple, fast, and green synthesis route of the particles. Another noteworthy fact is that the high penetration percentage was achieved without using any mucolytic agents, such as N-acetylcysteine, that result in physical and chemical disruption of the mucus layer [9]. Consequently, an effective drug delivery system was engineered completely step-by-step. The results of this study were highly promising in terms of developing nanocarriers that would be used in pulmonary drug delivery, not only for lung diseases, but also for any others in which the treatment is applied through the lungs.

and surface charge [13,21–25]. As the charge of the mucin network is negative [26], the surface charge of the drug delivery system becomes a major parameter, since negatively-charged vehicles will eventually lead to repulsion between the mucin and particle, and the positive surface charge will lead the attraction forces to dominate. In the literature, different strategies have been developed in order to protect the drug carriers from both the macrophages and clearance mechanism, and to enhance the penetration of drugs through the mucus layer. These include the encapsulation of the drugs in synthetic or natural degradable polymeric particles/capsules, stealth design of the particles, and use of liposomal delivery systems [27–31]. Mostly, the surfaces of the particles within a size range of 100–500 nm are modified with non-ionic surfactants, carboxyl, or amine groups, which provide the opportunity to study with particles possessing different surface charges and sizes [32]. It has been reported that particles of 500 nm or greater in diameter were immobilized within the network, whereas smaller particles of 100–300 nm were able to diffuse through this layer [20,21,24]. However, the penetration behavior of drug delivery systems through the mucus layer requires further enhancement [17,33]. Another main approach in the literature was designing virus-like muco-inert structures in terms of surface properties. As many viruses are able to cross the mucus barrier, it was proposed that particles coated with high-density cationic and anionic surface charge groups exhibited strong hydrophilicity, and an overall neutral charge allowed them to avoid adhesion within the mucosal network [5,34,35]. Muco-inert particles do not interact significantly with the mucus and they can diffuse through the pores in the mucus gel. To obtain this kind of drug carriers, synthetic particles are mainly used [24,34,36]. However, it is difficult to modify the surfaces of these particles so that they will be densely and equally covered with positive and negative charges, and obtain a net neutral surface charge. Even if such a surface modification were implemented, there would still be the risk that the particles would come up against the immune system while they transported through the mucus layer [5]. Moreover, there have been some concerns about the toxicity and removal of the organic phase during the synthesis of polymeric particles. As summarized, there are plenty of studies that aim to enhance the penetration behaviors of the particles via surface modification with different ligands, and therefore additional procedures should be applied to the particles after synthesis using additional chemicals, which would result in several drawbacks. On the other hand, lipid-based nanocarriers possess many advantages for drug delivery through the lungs. For example, if biodegradable and nontoxic lipids are chosen as the lipid phase, these carriers can be tolerated well in the airways and in the systemic circulation. In addition, their sizes can be modified so that the particles can be applied via aerosols. Moreover, surface modification can be implemented easily and thus, penetration of this kind of particles can be enhanced [37]. Generally, inhalation formulations that are used for the treatment of chronic lung diseases are applied at least 2 times in-a-day. Due to the fact that most lipid-based NPs provide prolonged release, the dosage of the applied drugs can be reduced, thus decreasing side-effects. Hence, for this, and many other reasons, the use of lipid-based NPs for drug delivery through the lungs is a convenient and necessary solution that still requires further development. Also, it has been reported in several studies that the cellular uptake of SLNs is favorable and efficient [38,39]. However, the mucus layer is the first barrier that has to be crossed before reaching the epithelial cells, so, interactions between the mucus and SLNs should be studied in detail before studying in vivo. In this study, mucus-penetrating solid lipid nanoparticles were prepared to obtain an effective drug delivery system. To understand the effect of the filtering mechanisms on the particles better, 3 different SLNs, possessing different size and zeta potentials, were prepared, and their biophysical interactions with the mucin solution were analyzed. Furthermore, their adsorption behaviors to the mucus layer were

2. Experimental section 2.1. Materials Herein, the stearic acid, Pluronic F127, and Tween 20, mucin from porcine type III, bound sialic acid 0.5%-1.5% (partially purified powder), bovine serum albumin (BSA), lecithin, polyacrylamide (PAA, molecular weight 5000–6000 kDa), sodium chloride (NaCl), sodium dihydrogen phosphate (NaH2PO4·H2O), disodium hydrogen phosphate dodecahydrate (NaH2PO4·12H2O), sodium azide (NaN3), calcium chloride (CaCl2), 3,6-bis(diethylamino)-9-(2-octadecyloxycarbonyl) phenyl chloride (R18), tetrahydrofuran (THF), and Tris were all purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA) and used without further purification. The water used in all of the experiments was ultrapure (UP) water with a resistivity of 18.3 MΩ-cm (MilliporeSigma, Burlington, MA, USA).

2.2. Methods 2.2.1. Preparation of mucin and CF solutions The pulmonary mucus model was mimicked using a 5% mucin solution (w/w), which was prepared in 0.9% NaCl and 0.01% NaN3 solution by magnetic stirring for 12 h at 300 rpm, as described previously [40]. It has been stated that pig mucus and human mucus are similar in structure and have a similar molecular weight [41,42]. Therefore, in order to obtain a standardized experimental system, pig gastric mucin was used throughout the experiments. A CF sputum model was mimicked by following a procedure described in detail elsewhere and by the references therein [42,43]. In brief, mucin, lecithin, and bovine serum albumin were dissolved in the buffer (85 mM Na+, 75 mM Cl- and 20 mM HEPES, pH 7.4) to achieve final concentrations of 60 mg/mL, 3.2 mg/mL and 32 mg/mL, respectively. Next, the resulting solution was magnetically stirred at 4 °C for 48 h. Both solutions were prepared freshly prior to the experiments, stored at 4 °C, and used within a week. 46

European Journal of Pharmaceutics and Biopharmaceutics 149 (2020) 45–57

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Table 1 Contents of the SLNs and the variation in the zeta potential values and sizes of the particles before and after incubation of the SLNs in the mucin solution for 2 h (a) SLN1, (b) SLN2 and (c) SLN3. Particle

SLN1 SLN2 SLN3

Content

Stearic acid Pluronic F127 Stearic acid Pluronic F127 Stearic acid Pluronic F127 Tween 20

Percentage (%)

3.3 2.5 3.3 2.5 3.3 0.8 1.7

Microemulsion/water ratio (v/v)

Zeta Potential Value (mV)

Size (nm)

Before incubation

After incubation

Before incubation

After incubation

1:20

−25.2 ± 3.2

−20.9 ± 2.2

100 ± 10

111 ± 8

1:10

−22.1 ± 2.1

−20.7 ± 1.9

35 ± 3

50 ± 4

1:20

−9.5 ± 1.4

−8.8 ± 1.2

90 ± 6

88 ± 10

∼122 cm2, with a subphase volume of ∼80 mL. The pulmonary mucus layer was mimicked by spreading the mucin solution (5% w/ v) on the air/buffer interface. The mucin layer was allowed to reach its specific equilibrium surface tension value for at least 20 min at the beginning of each experiment. The characteristic biophysical properties and hysteresis behavior of the equilibrated layer were obtained by continuously compressing and expanding the interface with Teflon barriers at a constant speed of 93.5 mm/min and at ambient temperature of 23 °C. For a better interpretation of the variations in the surface properties when particles and mucin/CF coexist at the interface, the characteristic interfacial properties of the SLNs were also determined by spreading the particle solutions to the air/buffer interface. For this purpose, SLN solutions were prepared at 2 different concentrations, 0.07 × 10-4 g/mL and 1.2 × 10-4 g/mL. After the injection of the particles to the air/ buffer interface, the variation in the surface pressure was recorded with time and therefore the adsorption ability of the particles to the air/ buffer interface was determined. Cyclic compression-expansion isotherms of the surfaces were also acquired after the adsorption of the particles to the interface. To analyze the interfacial behaviors of the NPs and mucin/CF, and their interactions with one another in a time-dependent manner, the variations in the surface pressures of the interfaces were also measured and recorded over time without disturbing the interface. To investigate the adsorption behavior of SLNs to the preformed mucin/CF layer, which had already equilibrated at a selected surface pressure (10 and 30 mN/m), SLN suspension was injected into the subphase of the mucin/CF layer as presented in Fig. 1b. The reproducibility was confirmed by repeating all of the measurements for at least 3 times. The standard errors for the measured interfacial pressures were determined as ± 2 mN/m. After the introduction of the particles beneath the previously compressed interface, variations in the surface pressure of the resulting interface were recorded over time and when the surface reached the steady state, the resulting interfaces were transferred to clean microscope slides via Langmuir-Blodgett technique for further analysis with AFM.

2.2.2. Preparation and characterization of the SLNs Preparation of the SLNs was performed via the well-known microemulsion method, by following the procedure as described in detail that was established previously by our group [44]. Briefly, the microemulsion was prepared by adding the lipid phase, which was composed of stearic acid at its melting point (∼70 °C), to the aqueous phase at the same temperature, which contained the chosen emulsifier dissolved in UP water. After the addition, the resulting mixture was magnetically stirred at 500 rpm for 10 min to form a transparent and thermodynamically stable microemulsion. The second step was the instantaneous solidification of microemulsion droplets by adding the hot microemulsion to cold water (2–4 °C) at a desired volume ratio. Properties of the particles used in this study, such as the components used in the preparation of the particles or microemulsion/water ratio, are summarized in Table 1. Further information about the characterization of the prepared SLN dispersions can be obtained from the relevant literature [44]. The size of the particles was determined via dynamic light scattering (DLS) (ALV-CGS-3, Malvern) analysis and AFM images which were acquired (PSIA Corporation, XE-100E) with Cr-Au cantilevers (ACTA 10 M) at a frequency of 0.37 Hz in non-contact mode. Zeta potential measurements were performed by applying ∼75 mV of potential difference with Zeta Meter System 3.0 (Zeta Meter Inc.) which consisted of a Quartz-Teflon GT-2 cell, molybdenum anode, and platinum cathode. Each measurement was performed for a minimum of three different batches. 2.2.3. Investigations of interactions between the SLNs and mucin 2.2.3.1. Interactions of the particles with mucin in solution form. To gain a deeper understanding of the interactions between the particles and mucin, particles were incubated in mucin solutions (which were prepared at two different concentrations: 0.5% w/w and 1% w/w) for 2 different time periods (2 and 8 h). After incubation, the suspensions were centrifuged at 6000 rpm for 20 min, and then washed with buffer 3 times to remove the non-interacted excess mucin. The degree of interaction between the particles and mucin was investigated by monitoring the changes in the size and the zeta potential of the particles.

2.2.4. In vitro penetration studies 2.2.4.1. Franz diffusion cell. Penetration performances of the particles were evaluated using the Franz diffusion cell, which has an effective diffusion area of 1.12 cm2. For the penetration studies, mucin solution was placed between the isopore polycarbonate membranes (Merck, Germany) with 400-nm mesh, which were attached to the ring located

2.2.3.2. Interactions of the particles with mucin/CF-mimicking surface at the air/buffer interface. Particle-mucin/CF interactions were studied further using a LB trough (Kibron, Finland) by creating a model of the pulmonary airways [45]. The trough had a surface area of

Fig. 1. Schematic representation for the 2-D investigation of the interactions of SLNs with mucin/CF layer at the air/buffer interface. (a) Compression of the mucin/CF layer to the desired surface pressure, (b) the injection of SLNs beneath the pre-equilibrated mucin/CF layer.

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3. Results and discussion

between the donor and receptor chambers. The membranes were soaked in water prior to the experiments. The penetration study with the Franz cell was implemented for 2 different mucin thicknesses, 2.6 ± 0.2 mm and 1.2 ± 0.2 mm. The donor compartment was loaded with particle suspension that was expected to penetrate through the mucus and the acceptor chamber was filled with fresh buffer only. The receptor site was magnetically stirred at 200 rpm during the experiment. After the addition of the NPs to the donor site, 400 μL of samples were withdrawn from the receptor chamber at different times (5, 15, 30, 60, 90, 120, 180, 240, and 300 min) and the same volume of fresh buffer was added to the compartment after each sampling. Penetration experiments were performed using rhodamine B-loaded SLNs to quantify the penetrated particle percentage. At the end of the experiments, the collected samples were treated with THF to disrupt the SLNs and provide the release of rhodamine B from the particles. The concentration of the penetrated particles was measured via colorimetric detection at 562 nm using a Biochrom EZ Read 400 ELISA microplate reader (Biochrom Ltd., UK). The penetration percentages and permeated amount of particles were calculated using the calibration curves elaborated at the same conditions. The effective diffusion coefficient (Deff) and permeability constant of the particles (Kp), were calculated according to the Fick’s law of diffusion and relevant equations were presented in Supporting Information.

3.1. Interactions of the SLNs with mucin in solution form It is known that most of the foreign particles are trapped in mucus layers via steric obstruction and adhesion [5]. So, the size and zeta potential of the NPs can affect their binding and transporting capabilities in mucus [33]. Particle-mucus interactions were first investigated via incubating the SLNs directly into the mucin solution. In this way, direct contact between the particles and the mucin was provided and from the differences of the zeta potentials, as well as the sizes of the particles before and after incubation, it became possible to observe if the mucin adhered to the particles [48]. This method provided preliminary data regarding the interactions between the particles and mucin as well as the prediction of the behavior of the particle after contacting with the mucus layer. To examine if there was an effect of the mucin concentration on the interactions, the mucin solution was prepared at 2 different concentrations. As a first step, the zeta potentials of the mucin solutions that were prepared in 1 mM NaCl were measured and were compatible with the literature (Table S1) [49]. The zeta potential measurements of the SLN suspensions were also implemented along with the size measurements obtained with the DLS and AFM. The zeta potential values of the SLNs in 1 mM NaCl solution were all lower than −30 mV, indicating that they were not strongly charged and were suitable for penetration studies through the mucus layer. The content of the SLNs was another important issue, since they were proposed to be used in drug delivery applications. As presented in Table 1, herein, stearic acid was chosen as the solid lipid phase for the preparation of the SLNs, since it is biocompatible and has been a generally preferred lipid in several studies [50–52]. The SLNs coded as SLN1 and SLN2 contained only Pluronic F127 as the emulsifier, whereas Tween 20 nonionic surfactant was also blended into the structure as a coemulsifier during the synthesis of the particle SLN3. Compatible with a previous study [44], the addition of the nonionic coemulsifier Tween 20 had decreased both the zeta potential and the size of the particle. The variations in the zeta potentials and sizes of the particles before and after incubating the particles in mucin solution are summarized in Table 1. Measurements were acquired after removing the nonadsorbed mucin from the solution. So, the effect of particle size on the interactions between the SLNs and the mucin had been also investigated in addition to zeta potential. To our knowledge, this was the first study that investigated the interactions of SLNs with mucin in such a parametric manner. According to the results, the zeta potential values of SLN1 and SLN2 had changed more than that of SLN3, showing that these particles had interacted with the mucin more than SLN3 had. The mucin concentration had no statistically significant effect on the final zeta potential values of the particles after incubation. The incubation period of the particles in 0.5% w/v mucin solution had been increased to 8 h and again the zeta potential values of the particles were determined as −21.5 ± 1.7, −20.3 ± 1.3, and −8.3 ± 1.1 mV for SLN1, SLN2 and SLN3, respectively, which were not very different than the results presented in Table 1. Further investigations were performed using the same samples and the results were supported by the size measurements implemented with AFM and DLS, which are presented in Table 1, Figs. S2 and S3, respectively. From the size measurements, the average sizes of SLN1, SLN2, and SLN3 before incubation were 100 ± 10, 35 ± 3, and 90 ± 6 nm respectively, whereas the final diameters after incubation were 111 ± 8, 50 ± 4, and 88 ± 10 nm for SLN1, SLN2, and SLN3. It was determined that even after incubating the particles in mucin solution, they were still able to redisperse in the solution and from the AFM images presented in Fig. S2, it was observed that they were monodisperse. As also seen in Fig. S2, the sizes of SLN1 and SLN2 were increased more than the size of SLN3. Even though the lipid content was the same for SLN1 and SLN2, due to the difference in their size, the surface charge

2.2.4.2. Rotating diffusion tubes. For further characterization of the penetration behavior of the particles through stagnant mucus, the rotating tube method was used [46]. For this purpose, silicon tubes (length: 45 mm; diameter: 3 mm) were filled with mucin solution [5% (w/v)] and the ends of the tubes were sealed with silicon caps. Rhodamine B-loaded SLN suspensions were introduced into one end of the tubes. After continuous rotation of the tubes at 30 rpm for different time intervals, such as 15, 60, and 240 min, the tubes were frozen at −80 °C overnight and cut into 15 slices of 3 mm each. Afterwards, the resulting sample at each segment was transferred to a clean microscope slide and analyzed with AFM. Particle distribution throughout the tube was calculated from the AFM images obtained from each segment of the tube using the Fiji Image J software. Therefore, the effect of the rotation time on the particle transport and final particle distribution through the tube was obtained. 2.2.5. Spreading of the particles on the mucus-mimicking subphase The spreading performances of the particles were evaluated using the LB trough and AFM on polyacrylamide gel, which is a commonly used subphase to mimic the pulmonary mucus layer [47]. Simply, 1% (w/w) solution (above the entanglement concentration) of PAA was prepared in 0.9% NaCl solution and stirred until the solution became homogeneous. The PAA solution was poured into a Petri dish and the surface pressure of the subphase was measured via the sensor of the LB trough, which was placed 1 cm away from the walls of the Petri dish. After the surface pressure of the subphase had reached steady state (approximately 20 min), SLN dispersions at 2 different particle amounts (3 × 10-3 mg and 12 × 10-3 mg) were applied to the center of the dish and the variations in the surface pressure were recorded over time. To enhance the spreading performance of the SLNs, after the surface pressure was equilibrated, 2.5 µL of catanionic surfactant solution of dodecyltrimethylammonium bromide (DTAB) and dioctyl sulfosuccinate sodium salt (AOT) mixed at xDTAB = 0.8 was also applied to the center of the Petri dish [40] and again, the surface pressure data were recorded over time, which allowed the observation of whether the particles were able to spread or not. The experimental method is represented in Fig. S1). Aside from the surface pressure measurements, the resulting interfaces, before and after the application of the catanionic solution, were transferred to clean microscope slides using the Langmuir-Schaefer method and visualized by AFM. 48

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density of the SLN2 (33 ± 3 × 1014 C/m2) was higher than the surface charge density of SLN1 (19 ± 0.6 × 1014 C/m2). This explains the reason why SLN2 had interacted with the mucin more than SLN1. On the other hand, there was no statistically significant increase in the diameter of the SLN3. The DLS results before and after incubation of the particles (Fig. S3) in mucin solution were compatible with the size measurements obtained from the AFM images, which also supported the results of the zeta potential measurements. Therefore, it can be concluded that among the 3 particle types, the one that had interacted the least with the mucin was SLN3. When particles contact with mucus, it is known that they are mainly influenced by repulsive and attractive forces such as electrostatic repulsion/ attraction. In the literature, it was reported that the penetration behavior of the particles enhanced when their zeta potential was close to zero, due to fewer interactions with the mucus [21,53]. For example, it has been stated that the diffusivity of PEG-coated NPs can be 3 orders of magnitude higher than the diffusivity of the uncoated NPs with a high zeta potential value [54]. Therefore, the results obtained from this part of the study were promising for carrying our further investigations.

was compressed to the chosen surface pressures via the barriers of the LB trough and the stability of the resulting layer on the interface was tested by measuring the surface pressure over time (Fig. S5). Here, the steady-state surface pressures of the compressed layers are important since after introducing SLNs through the subphase, the final surface pressure value will be one of the indicators for detecting the presence of the particles at the interface. As is seen in Fig. S5, when the layer was compressed to 10 mN/m, mucin monomers kept adsorbing to the interface for nearly 750 s, until the surface pressure reached 15 mN/m, which was the specific steady-state pressure of the noncompressed layer that had been determined previously (Fig. S5). However, when the layer was compressed to 30 mN/m, mucin monomers had started to desorb from the interface and the layer became stable at a surface pressure of 24 mN/m after 400 s. 3.2.2. Determination of the interfacial properties of the SLNs Following the characterization of the mucin layer at different conditions, the interfacial properties of 3 different SLNs were also characterized using different amounts of particles (Fig. S6). When developing a mucus-penetrating system, the surface characteristics of the particles are important. Therefore, it was thought that the adsorption behavior of the particles to the air/NaCl interface would provide a clue about their penetration behavior when the mucin layer is present at the interface. As is seen from the surface pressure-time isotherms for all of the particles in Fig. S6, the surface pressure had increased immediately after the injection of the particles from the subphase, independent of the particle concentration. The increment at the surface pressure proved that particles were able to adsorb to the interface. The adsorption behaviors of SLN1 and SLN2 were similar, as their lipid content was the same. On the other hand, SLN3 contained an additional surfactant (Tween 20) that had changed its surface properties, such as the zeta potential value, and thus its adsorption ability, as seen from its adsorption profile. For low particle concentration, the surface pressure of the interface had been increased in a fast manner and reached the steady state at 10.5, 11.6, and 19.1 mN/m for SLN1, SLN2, and SLN3, respectively. When the particle concentration was increased (from 0.07 × 10-4 to 1.20 × 10-4 g/mL), the steady-state surface pressures of the air/NaCl interface had increased from approximately 11 to 26 mN/m for SLN1 and SLN2, whereas for SLN3, it had increased from 20 to 35 mN/m. The hysteresis behavior of the interfaces after the injection of the particles into the subphase was also examined by compressing-expanding the interface at a constant speed (Fig. S7). From the compression-expansion cycles, it was determined that, for the conditions at which the SLN3 dispersion was injected into the subphase, the particle loss from the interface was less than that for the conditions, at which the SLN1 or SLN2 dispersion was injected beneath the interface. Furthermore, it was determined that the interface had reached higher surface pressures at the same surface area when SLN3 was present at the interface, which supported the result that the amount of SLN3 adsorbed to the interface was more than that of SLN1 and SLN2. Thus, the cyclic behaviors of the interfaces after the particle adsorption were also obtained as consistent with the previous findings of the study, where SLN1 and SLN2 behaved similar at the interface, whereas SLN3 could increase the surface pressure up to 42 mN/m due to its hydrophobicity.

3.2. Interactions of the particles with mucin at the air/mucin interface Particle-mucin interactions were also studied in 2D via analyzing the physicochemical changes in the air/mucin interface by measuring the variations in the surface pressure over time and changing the surface area. This method provides a convenient and useful way to study the properties of the thin films that spread over the aqueous subphases, similar to the airway mucus layer [45]. In this study, this method was used to develop a deeper understanding of the interactions between the particles and mucin layer and gain information about the penetration abilities of the particles. In the literature, several different methods have been used for modeling particle penetration through mucus, such as multiple particle tracking (MPT) or fluorescence recovery after photobleaching (FRAP) [5,20,55–57]. However, the mucus layer has a heterogeneous structure and even though this heterogeneity can be neglected at small-scale levels when the diffusion time and distance is increased, it may show an adverse effect on the diffusion behaviors of particles. It has been suggested that a 2D mucus model may overcome these drawbacks and decrease the experimental variability and support the findings obtained with other penetration methods [45]. The results of this section enabled the detection of whether the particles were able to adsorb to the mucin layer, diffuse through it, and integrate to the air/mucin interface. To understand if the particles can adsorb or trigger a change at the interfacial behaviors of the mucus layer, first, the interfacial properties of the mucin layer and SLNs were obtained individually, then the particles were introduced beneath the preequilibrated mucin layer. 3.2.1. Determination of the interfacial properties of the mucin at the air/ NaCl solution interface In order to mimic the pulmonary mucus layer, the mucin solution was spread on the air/NaCl solution interface and the steady-state surface pressure of the resulting interface was determined as 15 mN/m (Fig. S4a). The cyclic compression-expansion behavior of this layer was obtained compatible with the literature (Fig. S4b) [58]. The hysteresis behavior of the mucin layer at the air/NaCl interface indicated that mucin formed a stable layer at the interface, which reached a surface pressure of 45 mN/m in each compression cycle. As the pressure of the interface was increased due to compression, there was no evidence of collapse at the interface. This was also determined by the hysteresis behavior of the mucin layer (Fig. S4b). Hence, it is possible that after compressing the interface to a surface pressure of 30 mN/m, a multilayered structure was obtained at the interface, which was also supported by the AFM images (Fig. 2c). To change the physical structure of the mucin layer and to be able to study at different surface pressures, the preequilibrated mucin layer

3.2.3. Adsorption of the SLNs to the air/mucin interface After characterization of the interfacial properties of the SLNs at the air/NaCl interface, the adsorption of NPs to a 2D mucin layer that was compressed to selected pressures had also been investigated. From both the steady-state surface pressures of the interfaces and the AFM images, whether or not the particles were present at the interface was determined. Thus, the preliminary results for the penetration studies were obtained. Fig. 2a shows the surface pressure-time isotherms of the interfaces after injecting the SLNs beneath the interface, which was previously 49

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Fig. 2. Representative surface pressure-time isotherms of the interfaces after injection of 1.20 × 10-4 g/ml of SLN1, SLN2, and SLN3 from the subphase of the previously compressed mucin layer to (a) 10 mN/m, (b) 30 mN/m. (c) AFM images of the interfaces at steady-state which were obtained after injecting the SLNs from the subphase of previously compressed mucin layer to 5 mN/m, 10 mN/m and 30 mN/m (Interfaces were transferred to clean microscope slides after they had reached the steady state surface pressure). Arrows indicate the injection moments.

which was again compatible with the literature, since it is known that steric obstruction and adhesion are the main parameters that affect the penetration [59]. When the mucin layer was compressed to a higher pressure (30 mN/ m), as is seen in Fig. 2b, the injections of SLN1 and SLN2 increased the steady-state surface pressure of the native mucin for ∼3 mN/m. Only the injection of SLN3 had caused a noteworthy increment (≈8 mN/m) at the surface pressure, which was considered as an indication of the penetration ability of the particles. In the literature, it was stated in many studies that, diffusion through the mucus was more successful for neutrally charged particles [5,18,21,60]. That is the reason why the results obtained with SLN3 were more promising. Among the 3 particles, its zeta potential value was closest to zero. The resulting interfaces after the equilibrium, either with or without the particle injection, were transported to a clean microscope slide and

compressed to 10 mN/m. The thickness of this layer was calculated to be 33 µm, which simulated the thickness of the mucus layer in healthy individuals [5]. As can be seen, the final surface pressure values after the injection of all of the particles was higher than the steady-state surface pressure of the native mucin. This indicated that the 3 different SLN types were all able to adsorb to the mucin layer and reached the interface by diffusing through the mucin layer. Once more, the behaviors of SLN1 and SLN2 had been similar. As the diameter of SLN1 was bigger than that of SLN2, it adsorbed slower to the interface than SLN2, yet the final surface pressures of both particles were the same. The difference at the adsorption times of 2 similar particles at different sizes was agreeable with the literature, since it was stated that as the particle size increases, the particles diffuse slower. These findings also indicated that the surface charge was a more dominant parameter than the particle size during the adsorption and interaction with the mucin layer,

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properties of the mucin layer were changing continuously; therefore, these isotherms allowed the observation of the effects of the presence of SLNs at the interface under varying conditions. This continuous movement at the interface had also allowed the assembly of an analogy to the mucociliary clearance mechanism of the airways. It can be seen in Fig. 3 that, when SLN1 and SLN2 were injected beneath the mucin layer, even though the compression-expansion profile of mucin was changed, the maximum pressure value shifted to smaller areas at each cycle. This indicated that there was particle loss from the interface during the compression stage. On the other hand, the hysteresis behavior of the layer containing SLN3 had been very promising since the maximum pressure of the interface had been increased after the particle injection. This behavior for SLN3 had been attributed to its hydrophobic nature. There had been nearly no particle loss from the interface between the compression-expansion cycles, showing that particles could remain at the interface during the constant movement of the mucin layer once they reach the interface. In addition, the recruitment index of the interface containing SLN3 had been better than the other interfaces containing SLN1 and SLN2. In brief, among the 3 different SLNs, it was deduced that the results obtained with SLN3 were more favorable in terms of obtaining less interaction with the mucin and developing a novel mucus-penetrating drug delivery system. Therefore, SLN3 was used for the rest of the study during the investigation of the adsorption of the particles to the CFmimicking mucus model and also for further penetration studies.

visualized via AFM for further investigation. It can be seen from the AFM images (Fig. 2c) that as the surface pressure was increased, the structure of the mucin layer had changed and the surface became denser. For example, at a surface pressure of 10 mN/m, the pores of the mucin network can be seen easily, whereas, at a surface pressure of 30 mN/m, it can be seen that the areas between the mucin network had decreased due to the compression of the layer at the interface. The AFM images obtained from the resulting interfaces after the injection of the particles into the subphase had also supported the findings obtained from the surface pressure-time isotherms, as presented in Fig. 2c. The images show that, for all of the surface pressures, thus, for the different thicknesses of the mucin layer, the particles were successfully integrated at the air/mucin interface in each case. z-scale of the images were also presented next to the corresponding image, which showed the height profile of the interface. According to the zscale, particles (white-colored) are presented at the higher part of the image indicating that they had been located above the mucin layer. Despite the high surface pressure value of the mucin network at the interface (30 mN/m), which meant that the interface was in a more condensed state and the mucin layer was thicker (∼110 µm), SLN3 had penetrated through the pores of the mucin layer and successfully adsorbed to the interface. The cyclic compression-expansion behavior of the interfaces formed when the SLNs were injected beneath the preequilibrated mucin layer was also obtained after the interfaces had reached steady state (Fig. 3). During the compression-expansion process, the thickness and physical

Fig. 3. Representative cyclic compression-expansion isotherms of the interfaces obtained after the injection of 1.20 × 10-4 g/ml of (a) SLN1, (b) SLN2 and (c) SLN3 beneath the pre-equilibrated mucin layer. (Isotherms were obtained after the interfaces had reached the steady-state surface pressure). 51

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Fig. 4. (a) Representative surface pressure-time isotherms of CF layer that was compressed to 30 mN/m and the resulting interface after the injection of SLN3 beneath the CF layer that was pre-compressed to 30 mN/m, (b) representative cyclic compression-expansion isotherms of CF layer and the resulting interface after the injection of SLN3 beneath the pre-compressed CF layer, (c1 and c2) AFM images obtained from different sections the interface showing the structure of the CF layer that was compressed to 30 mN/m, c3) AFM image showing the structure of the interface after the injection of SLN3 beneath the CF layer that was pre-compressed to 30 mN/m. (Interfaces were transferred to clean microscope slides after the interfaces had reached the steady-state surface pressure).

of only CF at the same surface area. Therefore, it can be said that the layer had been fluidized and became physically more advantageous for drug delivery. Moreover, a plateau aroused during the compression. This plateau indicated the phase transition region and was an indicator of the presence of the lipid-based particles and proteins at the interface. The hysteresis showed that, after the introduction of the particles, the material loss from the interface had been decreased. This behavior can be attributed to the lipid-based particle-protein interaction at the interface [61]. Therefore, additional findings obtained in this section also demonstrated the penetration ability of the particles and usability of SLNs for drug delivery through the lungs, even in case of pulmonary diseases such as CF. The AFM images also supported the results obtained via the LB trough. It is known that the pore size of the mucin network in a healthy human is 400 nm on average. However, this value decreases to nearly 140 nm during CF [62]. As seen in the AFM images obtained by transferring the surface composed of the previously compressed CF layer, the pores had nearly coalesced and the layer at the surface became very dense and hard to diffuse through. Even in that case, it can be clearly seen from Fig. 4c3 that the particles had been successfully transported to the surface.

3.2.4. Adsorption of SLN3 to the air/CF-mimicking mucus interface The adsorption ability of SLN3 to the CF-mimicking mucus model was also investigated to examine whether the particles would be able to reach the interface even if a thick CF-mimicking mucus layer existed at the interface. In fact, this study was the first to investigate the adsorption behavior of SLNs to CF-mimicking mucus. In Fig. 4a, it can be seen that the surface pressure had reached steady state at ∼26 mN/m. After injecting SLN3 into the subphase of the previously compressed CF layer, it was determined that adsorption of the particles to the interface had resulted in an increase in the surface pressure of the interface up to 29 mN/m. In fact, this was a very critical finding since it showed that SLN3 was able to penetrate through this problematic layer containing additional components, such as lecithin and BSA, which changed the physicochemical properties of the mucin. Fig. 4b displays the hysteresis behaviors of both the CF-mimicking layer and the resulting interface after the injection of SLN3 beneath the CF-mimicking layer, which had previously been compressed to 30 mN/ m. The effect of the presence of SLN3 on the interface can be seen easily from the compression-expansion isotherms. During compression, the maximum pressure for the layer composed of only CF was determined as 46 mN/m. When the particles were injected beneath the interface, the maximum pressure had increased up to nearly 62 mN/m, which demonstrated the presence of the particles at the interface. As a result of the presence of SLNs at the interface, the surface had reached higher surface pressures when compared to the layer composed 52

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As can be seen in Fig. 5a, when both compartments were stirred, the percentage of penetrated particles was more (31.7%) than when only the receptor compartment was stirred (27.8%). This difference was an indication of a slight concentration polarization over the membrane. Even though the agitation speed in the donor compartment was relatively slower than that in the receptor compartment, it had homogenized the particle solution and prevented the polarization. Nevertheless, the setup where only the receptor compartment was stirred was considered more appropriate for mimicry of the airways. For this system, using both Fig. 5a and the inset figure, which show the variation in the cumulative amount of penetrated particles over time, it was determined that penetration reached equilibrium after 3 h. It was also verified that the particles did not decompose, lose their form, or release rhodamine B during the penetration by analyzing the samples taken from the receptor compartment of the Franz cell with AFM (Fig. 5b). The slope of the curve until the 3rd h was steep, which indicated that penetration was fast. Using the curve showing the cumulative amount of penetrated particles over time, Fick’s law of diffusion and the relevant equations in Supporting Information, the effective diffusion coefficient and permeability constant for the particles was calculated as 2.03 × 10-5 cm2/s and 1.08 × 10-1 cm/h. The diffusion coefficient and permeability constant of SLN3 were higher than the particles used in the literature, since the effective diffusion coefficient of different types of particles were reported to be in the range of 10-6–10-8 cm2/s [42,64]. In another study, the effective diffusion coefficient for Pluronic F127modified NPs was determined to be in the range of 10-8 cm2/s [65]. When only the receptor chamber was stirred, it was determined that 27.8% of the SLNs penetrated through the mucus after 5 h. In the literature, there are a variety of studies regarding obtaining mucus-penetrating drug delivery systems. In one study, the penetrated percent of

3.3. In vitro penetration studies with Franz diffusion cell & rotating diffusion tube After obtaining promising preliminary results for the penetration of the SLNs via the LB trough, further penetration studies were evaluated with the Franz diffusion cell and rotating diffusion tube methods. To be able to track the particles and quantify the amount of particle penetrated, rhodamine B was encapsulated in SLN3 during the synthesis, as described in the Experimental Section. In this way, rhodamine B had not only been used as a fluorescent probe, but it was also used as a model drug. The characterization of the rhodamine B-encapsulated SLNs was implemented via DLS and AFM, as shown in Fig. S8. The size of the NPs was obtained approximately as 170 ± 10 nm after the encapsulation and the zeta potential value was measured as −11.6 ± 0.8 mV, which was nearly the same value before the encapsulation. From both the increment in the size of the particles and the negative zeta potential value, it was concluded that rhodamine B was successfully integrated into the structure. It was also verified that the particles did not release the encapsulated dye unless they were disrupted with a chemical agent, such as THF. It is widely known that drug penetration through membranes such as skin can be easily simulated via the Franz cell, which is an FDAsuggested method [63]. Therefore, the in vitro diffusion behavior of SLN3 was also investigated with this method, by which airways can be modeled in 3D. During the penetration experiments, 2 parallel experiments were conducted, consisting of the agitation of only the receptor chamber or agitation of both compartments (donor and receptor) to detect the effect of possible concentration polarization, which can be considered a disadvantage of using a Franz cell. Fig. 5 shows the variation in the penetration percentages of the particles for 2 different setups.

Fig. 5. (a) Cumulative penetration percentage of particles diffused through the mucin layer, (Inset figure displays the cumulative particle amount varying with time during penetration) (b) AFM images of the sample taken from the receptor compartment of the Franz cell at the end of the 3rd hour (scale of upper image: 10 × 10 µm, scale of lower image: 5 × 5 µm). 53

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papain-modified NPs, which were 200 nm in diameter and had a −10 mV of zeta potential, were obtained as 9% [46]. In another study, the penetration percentage of self-emulsifying nanocarriers through a 280 μm-thick mucus layer was reported as 40%, even though the carriers consisted of mucolytic enzymes that were able to disrupt the mucus layer [66]. Moreover, another study reported that 35% of PEGcoated polystyrene particles could penetrate through a 10-μm-thick CF mucus layer. The penetration percentage increased to 75% only after disrupting the CF mucus layer via applying N-acetyl-cysteine [9]. On the other hand, in this study, lipid-based biodegradable and biocompatible NPs were used without applying any additional surface modifications. Also, there was no need for the addition of a mucusdisrupting agent to increase the mobility of the particles within this work. Thus, this penetration percentage was noteworthy when the mucus thickness between the polycarbonate membranes was considered. Reaching this penetration percentage showed that SLN3 was a mucus-penetrating drug carrier that can be used for drug delivery through the lungs. The effect of the thickness of the mucin layer on the penetration behavior of the particles was also investigated by decreasing the mucin thickness to approximately 1.2 ± 0.2 mm. The results were given comparatively with the previous results, in which the mucin thickness was 2.6 ± 0.2 mm (Fig. 6). As expected, the penetration percentages of the particles were increased when the thickness of the mucin layer between the membranes was decreased. Due to the decreased thickness, the particles had penetrated faster, as can be observed in Fig. 6. The final penetration percentage of SLN3 had reached up to 36% after decreasing the thickness. As 1.2 ± 0.2 mm was still a very high value when compared to the mucus thickness at the trachea or bronchus airways, where the average thickness of the mucus is 30 μm, reaching this percentage demonstrated that a mucus-penetrating drug delivery system had been obtained successfully. Moreover, it was already shown that the particles were able to diffuse through a thinner mucin layer, with the results established via the LB trough in the first part of the study. In addition to diffusion studies implemented with a Franz diffusion cell, the penetration ability of SLN3 through the mucus was also evaluated via the rotating diffusion tube method, which is also considered to be another method to study penetration [46]. With this method, diffusivity and mobility of the SLNs in bulk mucus were investigated in a parametric manner. The particle percentages at each segment of the diffusion tube, after rotating the tube for different time periods, are presented in Fig. 7.

Fig. 7. Percentages of particles determined at each segment of diffusion tube after rotation for 15, 60 and 240 min.

For the rotation period of 15 min, it was determined that 21% of the particles remained within the first 3 mm part of the tube, while 37.9% of the particles had accumulated in the middle section of the tube, between the 5th and 10th segment. Even though 15 min was a short time, it can be seen that particles were able to reach down into the last segment of the tube, which can be considered a successful result, showing the ability of the particles to diffuse in the mucus when compared with the literature [46]. To observe the effect of the rotation time on the diffusion distance of the particles, the rotation period was increased to 60 min. After 60 min of rotation, the particle percentage in the first segment had decreased to 14%, while that in the middle section had increased to 49.4%, showing that the increase in the rotation period resulted in an increase in the percentages of the penetrated particles. Moreover, this result indicated that the particles did not stick in the mucin network. Increasing the rotation time to 240 min had decreased the particle percentage in the first segment down to 4.8%. Thus, nearly all of the particles were able to diffuse through the tube. The particle percentage in the last segment had increased by 3-fold; from 1.1% and 1.4% for 15 and 60 min of rotation to 3.22% after 240 min. The local maximum particle amount was reached in the 7th segment as in the case for 60 min of rotation. Moreover, the particle amount had increased to 55.9% and 24.2% in the middle and end sections (the last 5 segments), respectively. Once more, it can be concluded that SLN3 was able to diffuse efficiently through the mucus layer and therefore it is a potential candidate to be used in drug delivery through the lungs.

3.4. Spreading of the particles on the mucus-mimicking subphase To obtain a complete and well-functioning drug delivery system, it is vital to transport a sufficient amount of drug to the targeted region. As it is important for the particles to penetrate through the mucus layer, it is also important for them to spread effectively and homogeneously on the pulmonary airways before penetration. From this point of view, in this study, for the first time in the literature, the spreading performance of the particles on the mucus-mimicking subphase was also investigated. The spreading experiments were conducted on entangled PAA gel to better observe the spreading of the particles on the surface, which is a generally preferred subphase to mimic the mucus layer [68]. The spreading abilities of the particles were analyzed by monitoring the variations in the surface pressure at 3.5 cm away from the center of the preequilibrated subphase on where injection of the SLNs at 2 different concentrations had been implemented (Fig. 8). As seen in Fig. 8a, after injection of the SLNs on the preequilibrated

Fig. 6. Cumulative penetration percentage of particles diffused through the mucin layer. 54

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Fig. 8. Surface pressure-time isotherms of the PAA subphase (a) after the injection of SLN3 suspensions on the center, (b) after the injection of SLN3 suspension and the catanionic surfactant mixture on the center. Arrows indicate the injection moments of the suspensions and catanionic mixture, (c) AFM images that were acquired from the points located at the center, 2 cm. away and 3 cm. away from the center before and after applying the catanionic surfactant mixture.

adequate surface tension difference that would trigger the spreading of the particles, the mixture of xDTAB = 0.8 was applied after introduction of SLN3 on the subphase (Fig. 8b). As seen in Fig. 8b, when the mixture with xDTAB = 0.8 was injected, the surface pressure had increased immediately to 46 mN/m, showing that the catanionic mixture was able to spread on the surface in a very fast manner. Moreover, the Marangoni-dominated spreading behavior of the mixture had enhanced the spreading of the SLNs, as shown by the surface pressure-time isotherms. After applying the catanionic mixture onto the center of the subphase where the SLNs were first introduced, again independent of the particle amount, the surface pressure had increased immediately to nearly 50 mN/m, which was a higher value than the surface pressure of the xDTAB = 0.8 mixture on the PAA subphase. As the increments in the surface pressure demonstrated an

PAA surface, the surface pressure had increased to 37 mN/m, independent of the particle amount. The steady-state surface pressure of the PAA subphase was 30 mN/m. Thus, it can be concluded that the particles did not possess an efficient spreading ability. As the dispersing agent of the particle suspension was water, the spreading ability of the applied solution was limited due to the high surface tension of water. It is known that the spreading of one phase onto another strongly depends on the surface tension difference between the two phases. In order to enhance the spreading ability of the SLN dispersion, it was decided to transport the particles using a superspreading solution that was composed of DTAB and AOT. In previous studies, it was demonstrated that the catanionic surfactant solutions of DTAB and AOT mixed at xDTAB = 0.8 provided an enhanced spreading behavior on the mucus subphase [40]. Therefore, for this part of the study, to obtain an 55

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Appendix A. Supplementary material

increase in the amount of material present at the interface, this result showed that the particles had been transported along with the surfactant mixture. The results were also supported by the AFM images acquired from the points that were located in the center, and 2 and 3 cm away from the center (Fig. 8c). When the images were compared for the two cases, where the surfactant mixture was either applied or not, it was seen that the density of the particles at the center had decreased after applying the surfactant mixture. It was also observed that the number of particles had increased significantly at the point 3 cm away from the center, proving that particles had been successfully spread with the surfactant mixture. Oppositely, when the surfactant solution was not applied on the surface, hardly any particles were detected at the point 3 cm away from the application point. Overall, for the first time in the literature, the spreading performance of SLNs was investigated, and even enhanced, using a catanionic surfactant mixture of DTAB and AOT that was composed of xDTAB = 0.8. As it is important to deliver the appropriate amount of drug to the targeted region, this part of the study holds a critical position for analyzing the full performance of a drug delivery formulation from the spreading step to penetration through the mucus layer.

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4. Conclusion Efficient transportation of drug delivery vehicles to the target site is the key parameter for enabling a successful drug delivery application. In the case of drug delivery through pulmonary airways, carriers should overcome the mucus layer to reach the epithelia. Although numerous approaches and strategies exist in the literature, a lipid-based system that is able to spread uniformly on the airways and penetrate through the pulmonary mucus with high penetration percentages could not be obtained without disrupting the layer. As the dominant mechanisms behind the poor penetration abilities of the particles are the size and interaction filtering of the mucus gel, these filtering mechanisms employed by the mucus should be understood well when developing mucus-penetrating particles. In this study, SLNs were used as model drug carriers and their spreading and penetration ability on and through the pulmonary mucus was studied in both 2D and 3D. From the surface pressure-time isotherms and hysteresis behaviors of the interfaces, preliminary results for the penetration abilities of the SLNs and their biophysical interactions with the mucin were obtained. Among the 3 different SLNs, SLN3 had interacted with the mucin less than the other particles due to its hydrophobicity and zeta potential value. Thus, when SLN3 was introduced beneath the mucin layer, it could adsorb to the mucin layer, penetrate through the pores, and reach and integrate into the interface more effectively than SLN1 and SLN2. As expected, the low interaction of SLN3 with the mucin provided high diffusivity. From the Franz diffusion cell method, it was obtained that nearly 28% of the particles could penetrate through the 2.6 ± 0.2 mm-thick mucus, whereas this percentage was increased to 36% when the thickness of the mucin layer between the membranes was decreased to 1.2 ± 0.2 mm. This study was the first to reach this penetration percentage with SLNs through a considerably thick mucus layer, without any modification on the surfaces of the particles or any chemical disruption of the mucus layer with an agent. As a conclusion, herein, it was shown that this newly-developed mucus-penetrating SLN formulation is appropriate to be tested in in vivo studies and to be used in further studies involving drug delivery applications through the lungs. Acknowledgments This work is supported by The Scientific and Technological Research Council of Turkey (TUBITAK) partially through Grant Numbers 215M759 and 113M259. The authors also would like to acknowledge Prof. Dr. Levent Yılmaz for his valuable discussion. 56

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