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Perfluorohexyloctane (F6H8) as a delivery agent for cyclosporine A in dry eye syndrome therapy – Langmuir monolayer study complemented with infrared nanospectroscopy
Chachaj-Brekiesz, Anna
Wnętrzak, Anita
Lipiec, Ewelina
Kobierski, Jan
Dynarowicz-Latka, Patrycja
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Colloids and Surfaces. B, Biointerfaces
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10.1016/j.colsurfb.2019.110564
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110564
110564
S0927-7765(19)30708-8
10.1016/j.colsurfb.2019.110564
The Authors
Fig. 1
π–A isotherms of DPPC:DPPE:SM mixed film without and in presence of F6H8 (a), Cs
−1 (at π = 15 and 28 mN/m) as function of F6H8 content (b).
Fig. 1
Fig. 2
AFM images of LB films DPPC/DPPE/SM (a) and DPPC/DPPE/SM with addition of 30 mol% F6H8(b).
Fig. 2
Fig. 3
π–A isotherms of DPPC/DPPE/SM system in the presence of CsA (a) or containing both CsA and F6H8 (b).
Fig. 3
Fig. 4
The effect of CsA and F6H8 on DPPC/DPPE/SM film: Cs
−1 in function of CsA content (a); ΔGexc (b). Values calculated at π = 15 mN/m.
Fig. 4
Fig. 5
AFM imaging of DPPC/DPPE/SM monolayer with addition of 30 mol% CsA (a); 30 mol% CsA and 30 mol% F6H8 (b); 30 mol% CsA and 50 mol% F6H8 (c).
Fig. 5
Fig. 6
Comparison of FTIR-ATR spectra of pure compounds in bulk (upper), DPPC/DPPE/SM and DPPC/DPPE/SM treated with CsA and F6H8 (middle) with AFM-IR spectra probed with p-polarization from DPPC/DPPE/SM film treated with CsA and F6H8 (lower).
Fig. 6
Fig. 7
AFM-IR spectra probed with different polarizations (s- and p-) from DPPC/DPPE/SM film treated with CsA and F6H8: inside (lower panel) and outside (upper panel) the domain together with molecular models.
Fig. 7
Fig. 8
PCA analysis of AFM-IR spectra collected with p-polarised light from the domains and from the neighbouring areas.
Fig. 8
Perfluorohexyloctane (F6H8) as a delivery agent for cyclosporine A in dry eye syndrome therapy – Langmuir monolayer study complemented with infrared nanospectroscopy
Anna
Chachaj-Brekiesz
a
*
chachaj@chemia.uj.edu.pl
Anita
Wnętrzak
a
Ewelina
Lipiec
b
c
Jan
Kobierski
d
Patrycja
Dynarowicz-Latka
a
a
Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland
Faculty of Chemistry
Jagiellonian University
Gronostajowa 2
Kraków
30-387
Poland
Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland
b
Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland
Faculty of Physics, Astronomy and Applied Computer Science
Jagiellonian University
Łojasiewicza 11
Kraków
30-348
Poland
Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland
c
The Henryk Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Kraków, Poland
The Henryk Niewodniczański Institute of Nuclear Physics
Polish Academy of Sciences
Radzikowskiego 152
Kraków
31-342
Poland
The Henryk Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Kraków, Poland
d
Department of Pharmaceutical Biophysics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland
Department of Pharmaceutical Biophysics
Faculty of Pharmacy
Jagiellonian University Medical College
Medyczna 9
Kraków
30-688
Poland
Department of Pharmaceutical Biophysics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland
⁎
Corresponding author.
Graphical abstract
Highlights
•
Mixed Langmuir monolayers were used to mimic polar lipids compartment of TTL.
•
CsA affects tear phospholipids assembly by loosening of molecular packing.
•
Effectiveness of F6H8 as delivery agent for CsA was confirmed.
•
Low-cost laboratory approach for membrane active drugs studies was proposed.
Abstract
One of the key challenges in dry eye syndrome therapy is to find a suitable carrier for immunosuppressant drug – cyclosporine A (CsA) – delivery to the eye. To investigate this issue, herein we present a methodology based on the combined analysis in macro- (Langmuir monolayers), micro- (Brewster angle microscopy) and nanoscale (atomic force microscopy and infrared nano-spectroscopy). The applied approach proves that CsA affects the phospholipid part of the tear film lipid layer by loosening molecular packing. This effect can be reversed by the addition of perfluorohexyloctane (F6H8). We have highlighted that F6H8 increases the availability of CsA and therefore is appropriate carrier for CsA topical delivery to the eye in the dry eye syndrome. In addition, the applied herein procedure provides a simple, low-cost laboratory tool for preliminary studies involving membrane active pharmaceuticals, preceding in vivo tests.
Abbreviations
AFM-IR
infrared nano-spectroscopy
ATR-FTIR
attenuated total reflection Fourier transform infrared spectroscopy
BAM
Brewster angle microscopy
CE
cholesteryl ester
CsA
cyclosporine A
DES
dry eye syndrome
DFT
density functional theory
DPPC
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DPPE
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine
F6H8
perfluorohexyloctane
LB
Langmuir-Blodgett
OAHFA
O-acyl-ω-hydroxyfatty acid
PCA
principal component analysis
SM
sphingomyelin
TLL
tear film lipid layer
WE
wax ester
Keywords
LB films
Tear film
Cyclosporine A
Semifluorinated alkanes
AFM-IR
1
Introduction
A disorder in the tear film homeostasis can lead to dry eye syndrome (DES), which is a multifactorial disease of the ocular surface [1]. The tear film stability is ensured by its three layers: mucine, aqueous and lipid. The latter, the tear film lipid layer, TLL, consists of amphiphilic sublayer covered with the external nonpolar film [2]. DES can occur as a result of deficiency of aqueous layer, excessive evaporation of tears (i.e. due to abnormalities in TLL) or both [3]. Epidemiological research has demonstrated that frequency of DES occurrence is within 4.4% to even 50% among middle-aged and elderly people worldwide [4]. Pathomechanism of this disease is complicated and diagnostics is multistage (starting from a detailed patient interview through simple laboratory tests, ending with lipidomic analysis of TLL). Therapy is focused on the recovery of tear film homeostasis and predominantly topical symptomatic treatment is applied [5]. In order to relieve symptoms beyond the limitation of environmental impact and eye hygiene improvement, the following treatments can be applied: artificial tears [6], TLL supplements [7] (e.g. castor oil [8], semifluorinated alkanes [9,10]), steroids [11], macrolidic antibiotics and tetracyclines [12].
Currently, DES is associated with inflammatory diseases very common in autoimmune disorders. Therefore, one of the most effective DES treatments is based on the topical use of cyclosporine A (CsA) – cyclic polypeptide, commonly known for its immunosuppressant properties [13]. Namely, at the receptor level, CsA acts as an inhibitor of interleukin-2 release during the T-cells activation and causes suppression of cell-mediated immune response. As a result, it increases both the production of tears and the density of goblet cells [14,15]. The effectiveness of CsA was confirmed by many biological experiments on animal models [16–18] and in clinical trials [19]. Nowadays, there are several commercially available ocular formulations based on CsA (for details, see review [20]).
Limitation of the abovementioned approach to DES therapy is associated with difficulties in CsA delivery to the eye. This is related to both the large molecular size of the peptide and its hydrophobicity. If a disorder occurs in the inner layers of the tear film, the therapeutic substance should be transferred through the outer TLL (consisting mostly of nonpolar lipids), and therefore improving the penetrating properties becomes crucial. This can be achieved, among others, by the selection of an appropriate delivery agent that increases CsA penetration into the eye. Such a delivery agent should be characterized – like CsA – by high hydrophobicity. In addition, it should be chemically inert to the tear film and remain neutral to the eye. Carriers for CsA, which are typically used in ocular formulations, are based on nonpolar lipids, such as castor, corn or olive oil and medium-chain triglycerides. Another applied strategy is to improve CsA solubility in ethanol with non-ionic surfactants, e.g. polyoxyl-40 or polysorbate 80 [20].
Promising delivery agents for CsA can be semifluorinated alkanes, which have been extensively studied in our laboratory [21,22]. These compounds are characterized by unique combination of apolarity and amphiphility. This allows for their easy distribution both in apolar and amphiphilic sublayer of TLL. Preliminary studies have shown that such unique combination of properties ensures CsA dissolution and transfer across apolar waxes to the amphiphilic sublayer of TLL. The results of model studies on animals using perfluorohexyloctane (F(CF2)6(CH2)8H, abbr. F6H8) have been found to be promising [23,24]. It should be emphasized that semifluorinated alkanes are neutral to ocular surface and themselves possess a therapeutic effect [9,10]. However, the detailed action mode of CsA and semifluorinated alkanes at the molecular level has not been clarified so far. Therefore this paper is aimed to fulfill this gap.
Disorder in biophysical properties of the tear film observed in dry eye syndrome may result from dysfunctions in amphiphilic sublayer of TLL [5], which - in the normal conditions - forms highly ordered system on the surface of aqueous layer and is isolated from the environment by the nonpolar film. Such systems can be characterized with the Langmuir monolayer technique, which was applied to investigate mechanic properties of films from natural samples of human and animal meibomian gland secretions alone [25,26] as well as treated with pharmaceuticals (i.e. hyaluronic acid [27], Cationorm® [28]), drug preservatives (benzalkonium chloride [27,29,30]) or peptides [31]. Some limitations of this approach should be mentioned, i.e. natural samples can be collected only in sub-milligram quantities (which is insufficient for Langmuir experiments); also they show variable composition depending on individual donors and sample collection protocol [31,32]. The main disadvantage is systematic error based on the assumption that the composition of the tear film lipid layer is reflected in the composition of meibomian gland fluid. As reviewed in [32], the lipid composition of meibum differs significantly from that of tears, especially in the phospholipids content (0.006-0.1 mol% in meibum and 5.4–90 mol% of tear lipids). Therefore amphiphilic phospholipids, which physiological origin remains unknown, should also be taken into consideration as they strikingly influence surface properties of films (for detailed analysis see [33,34]) in contrast to nonpolar lipids [35]. Phospholipid/nonpolar lipids mixtures [33,36,37] as well as phospholipids alone [38–40] were successfully applied as artificial models mimicking tear film lipid layer. Model approach allows to focus on a selected part of the tear film (amphiphilic or apolar sublayer of TLL) and monitor the incorporation of surface active agents. Well-defined system allows to monitor rheological properties of samples and additionally describe molecular interactions.
To obtain a complex picture of i) molecular organization, ii) variations in surface activity, and iii) thermodynamics of the artificial lipid layer of the tear film in the presence of CsA and F6H8, we decided to carry out an analysis in macro- (thermodynamics of interactions based on the Langmuir monolayer technique), micro- (Brewster angle microscopy, BAM) and nanoscale (atomic force microscopy, AFM and infrared nano-spectroscopy, AFM-IR). As a simplified model of the TLL we considered a mixture of phospholipids, i.e. DPPC:DPPE:SM (in proportion 3:2:2) as it reflects their proportions in natural tears [31,41]. It is worth mentioning that the exact lipid composition of natural TLL still remains unclear, however, apart from phospholipids also O-acyl-ω-hydroxyfatty acids (OAHFAs) and high proportion of nonpolar lipids, such as cholesteryl esters (CEs) and wax esters (WEs) are present [33]. When modeling the TLL we did not take into account nonpolar lipids since stability of the tear film is related to the surface activity of the lipid layer [33]. CEs and WEs have been found to be of low surface activity [42], and although at low surface pressures they can coexist together with phospholipids in the form of a monolayer, at high pressures (corresponding to physiological conditions) they are expelled from the monolayer, forming an overlaying layer [33]. Therefore they do not contribute in any significant way to the surface activity of the lipid layer. The role of OAHFAs in TLL has long been unknown until recent report [43] showing their protective role against evaporation of the tear film, however, their surface activity remains low, similarly to CEs and WEs. Taking all the above into consideration and given that we were interested in receiving a stable system which allows monitoring changes in surface activity and conducting thermodynamic analysis, system composed of amphiphilic phospholipids seems appropriate as a simplified model of the tear film lipid layer.
The results of our experiments allowed to understand the mechanisms responsible for CsA and F6H8 therapeutic activity (especially the F6H8 role as a delivery agent). Moreover, our methodology can be found useful for further investigations of properties and stability of tear film lipid layer as a part of preliminary research preceding in vivo tests.
2
Results and discussion
2.1
Langmuir monolayer study completed with BAM and AFM imaging
Measurements of surface pressure−area (π–A) isotherms are a classical and conventional way of characterizing monolayers stability, phase behavior of molecules in Langmuir films, intermolecular interactions and miscibility between components.
To be able to relate the obtained results to physiological conditions, the experiments should be performed at high surface pressure region (26–30 mN/m [39]) and at temperature of 37 °C. Unfortunately, we could not meet the pressure requirement for film containing CsA and had to conduct experiments (analysis of interactions and floating layers textures as well as the transfer of the films onto a solid substrate) at 15 mN/m, as it was the highest pressure for which the monolayer containing CsA was stable. Due to technical difficulties (high water evaporation at elevated temperatures, disturbances in the operation of electronics and microscope) the experiments were performed at room temperature (20 °C). F6H8 alone is not capable for monolayers formation and therefore was excluded from considerations regarding the influence of temperature, CsA was found not to be much sensitive to the change of temperature within the range of 20–30 °C (Fig. S.1 in Appendix A), contrary to phospholipids. However, their surface activity remains high both at lower (20 °C) and higher (above 30 °C) temperature. This proves that temperature plays a minor role in terms of surface activity. Moreover, tear lipid layer needs to maintain its functions within the range of ambient temperatures (20–30 °C) [34], and therefore the exact value of experimental temperature is of low impact in the analysis of our results.
Firstly, in order to check the effect of F6H8 on monolayer mimicking TLL, we recorded π–A isotherms for respective multicomponent films with and without F6H8 (in proportion of 10, 30 and 50 mol%) (Fig. 1
).
The π–A isotherm for phospholipid mixture starts to rise at about 73 Å2/molecule. Upon compression, the surface pressure rises homogeneously until film collapse, which occurs at about 70 mN/m. The additio n of F6H8 does not influence isotherms’ shape, however, they become shifted toward smaller areas in respect to the model. Interestingly, the physical states of the investigated monolayers, reflected in compression moduli (Cs
−1 =
-
A
d
π
d
A
[44]) values, are similar (Cs
−1 ≈ 90 mN/m for π = 15 mN/m), regardless of the presence and amount of F6H8 (see inset in Fig. 1). At 28 mN/m (which is in the middle of the natural tear film surface pressure range [39]) the addition of F6H8 slightly increases Cs
−1 values (the increase is inversely proportional to F6H8 content). All the investigated films fall within liquid state. BAM images obtained for systems with different content of F6H8 (Fig. S.2 in Appendix A) show almost the same texture of monolayer (oval domains).
In the next step, the investigated monolayers were transferred onto mica and their topography was recorded with AFM. Careful analysis of AFM images acquired from mixed phospholipid system alone and with addition of F6H8 (Fig. 2
) indicated that both studied surfaces are homogenous and smooth. Profiles extracted along lines marked on AFM images are comparable. These confirms that addition of F6H8 does not affect the smoothness and flatness of the surface.
In the next step we have checked changes induced to investigated monolayer by the addition of cyclosporine A. Firstly, CsA in two different proportions (10 and 30 mol%) was added (Fig. 3
a). Then, F6H8 (30 and 50 mol%, respectively) was added to the system with 30 mol% content of CsA (Fig. 3b).
In mixed phospholipids/CsA systems (Fig. 3a) the presence of CsA causes shifting of the isotherms towards larger areas per molecule, which indicates an increase of intermolecular spacing upon addition of CsA. The slope of the curves is also different in comparison to reference (DPPC/DPPE/SM). In the curve recorded for the system with 10 mol% content of CsA, two collapses are visible: one corresponds exactly to the πcoll for pure CsA, and the other one occurs at a higher pressure. Unfortunately, it is impossible to precisely define its value due to its occurrence at a low area (which is out of the moving barrier range). In general, the presence of more than one collapse in the course of an isotherm formed by different components evidences for their immiscibility (total or partial) in a monolayer. For mixtures with 30 mol% content of CsA one can also expect two collapses, however, the lack of second collapse results from the same technical reasons as mentioned above.
The addition of F6H8 to the monolayer containing 30 mol% of CsA (Fig. 3b) causes that the isotherms are slightly shifted towards smaller areas per molecule in comparison to the curve registered for the system without F6H8. The shift depends on the content of F6H8 - the larger the addition is, the smaller area per molecule. Moreover, isotherms for systems containing F6H8 are slightly steeper. In the course of these isotherms only one collapse is observed (this may also be due to technical limitations).
In order to provide a detailed description of the effect of CsA and F6H8 on the investigated phospholipid mixture, Cs
−1 values were calculated (at 15 mN/m) and can be seen in Fig. 4
a.
The pure DPPC/DPPE/SM mixture and CsA individually form monolayers of the liquid state. The incorporation of CsA causes a decrease in film compressibility moduli, especially visible for monolayer with 30% content of CsA. After F6H8 addition, Cs
−1 values increase. It can be connected with the change of molecular ordering in the film.
In Fig. S.3 (see Appendix A) a comparison of BAM images registered at 15 mN/m for pure phospholipid mixture and systems treated with different amount of CsA (with and without presence of F6H8) have been shown. As it can be seen, film textures change significantly after CsA incorporation. Domains characteristic for DPPC/DPPE/SM system disappear: only grains with insignificant, blurred borders are observed. The addition of F6H8 to the film containing 30 mol% of CsA again changes the texture. For the monolayer poor in F6H8, circular domains are observed, while in mixture with high content of F6H8, BAM images are more homogenous, however, the texture is not smooth. To analyze the structure of the investigated films more deeply, selected systems were transferred onto mica and AFM topographies were recorded.
Fig. 5
demonstrates AFM images of DPPC/DPPE/SM monolayer with addition of pure CsA (Fig. 5a) and CsA with two different doses of F6H8 (Fig. 5b and c). As it can be seen, CsA induces domains formation. In each topography, 1 nm height domains (see profiles) were detected. However, the lateral size of domains was determined by a content of F6H8. Our results indicate that an addition of 50 mol% F6H8 causes fragmentation of large (4–6 μm diameter) domains induced upon CsA incorporation.
To better understand the role of CsA and F6H8, the excess free energy changes (ΔGexc) have been determined (see Fig. 4b). ΔGexc were calculated from the formula
Δ
G
e
x
c
=
N
A
∫
A
12
-
A
1
X
1
+
A
2
X
2
d
π
[45], where A1
is mean molecular area of reference system (DPPC/DPPE/SM with or without addition of F6H8), A2
is mean molecular area of CsA and A12
is mean molecular area for DPPC/DPPE/SM film with CsA (with or without addition of F6H8), X2
is molar fraction of CsA and X1
is molar fraction of reference system (X1 = 1-X2
). Analysis of the results shows that F6H8 influences mutual interactions between CsA and investigated phospholipids mixture. Initially, for the film containing CsA, ΔGexc value is positive, indicating more repulsive interactions between phospholipids and CsA in comparison to reference system (DPPC/DPPE/SM). Positive values may also point to a lower stability of this mixed system. In turn, F6H8 stabilizes the system (ΔGexc is negative), indicating that interactions become more attractive (or less repulsive) in comparison to interactions in abovementioned monolayer.
The above-described approach (Langmuir monolayer analysis combined with BAM and AFM) does not provide sufficient information on the composition of the separated phases, therefore further characterization involving nano-spectroscopic method was performed.
2.2
Spectroscopic results
2.2.1
IR spectra in nanoscale compared to bulk samples
To determine chemical composition of different phases in sample of DPPC/DPPE/SM film treated with pharmaceuticals (CsA and F6H8), AFM-IR technique [46,47] was applied. Single, floating layers were transferred onto gold coated silica and infrared spectra (in the spectral range 1150–1900 cm−1) were probed at the nanoscale. Places of spectra acquisition were selected based on AFM topography (phase inside and outside domains). Course of representative spectra collected with p polarization was presented in Fig. 6
. As it can be seen, spectra recorded from similar places of sample are analogical to each other. Meanwhile, signal from regions localized in domains is different in comparison to that probed from surrounding phase. This suggests differences in chemical composition within and outside domains. At the same time, distribution within each phase is suggested to remain homogeneous. The homogeneity issue will be characterized with PCA tool (see paragraph 2.2.3).
Further analysis of experimental results obtained from nanoscale required comparison with ATR-FTIR spectra collected from bulk samples. Initially, ATR-FTIR spectra of DPPC/DPPE/SM mixture and DPPC/DPPE/SM treated with CsA and F6H8 were compared to AFM-IR spectra (Fig. 6). As it can be seen spectra from nanoscale contain similar bands to those from mixtures in bulk. Differences in mutual intensities of selected bands (nanoscale vs bulk) indicate that monolayer samples are more ordered and molecular distribution is not random.
Finally, AFM-IR spectra registered with p polarization were systematically compared to ATR-FTIR spectra of bulk substances to notice some important remarks on chemical composition of each phase. Moreover IR spectra of the studied compounds were calculated (for details see Appendix A) and vibrational assignments of observed and calculated frequencies were presented in Table S.1 (Appendix A). This enabled us to select spectral markers of each compound.
In investigated spectral region F6H8 absorption is poor with exception of rather wide band with maxima at 1188 and 1234 cm−1 attributed to stretching (ν(C–F), ν(C–C)) and bending (wagging ω(C–H), rocking ρ(C–H), twisting τ(CH)) vibrations. This band is similarly noticeable in spectra probed in nanoscale from inside and surrounding of the domain. Its intensity is almost independent from place of data acquisition therefore it allows to conclude that semifluorinated alkane constitutes matrix for other molecules in film. Polypeptide cyclosporine A in IR spectra shows characteristic band from CH3 deformational vibrations (at 1410 cm−1), as well as wide intensive bands attributed to amide II (at 1540 cm−1) and amide I (in region 1600–1650 cm−1). Bands observed in nanoscale at 1410 and 1540 cm−1 are more intensive and characteristic to spectra collected inside the domains. The increase of intensity of band at 1600–1650 cm−1 outside of the domain seems to be confusing, however it can be explained by overlapping with the amide I band characteristic for SM (which concentration is probably greater outside domains). Therefore it can be concluded that cyclosporine A is preferentially accumulated within oval domains. To determine distribution of phospholipids with ester moiety (DPPE and DPPC) diagnostic bands 1702 and 1744 cm−1 (arising from ester moieties) were selected. Those bands are more intensive outside domains therefore surrounding phase is enriched in phospholipids.
In the analyzed spectral region there are also bands which originate from all studied compounds and consequently should not be taken into consideration when looking for spectral markers of compounds distribution, however they can suggest degree of molecular packing and ordering. For example, intensive band with maximum at 1480 cm−1 is characteristic for all phases of sample as it arises from cumulated molecular vibrations of hydrocarbon chains. This band is narrower in case of spectra probed inside the domain therefore it suggests the increase of molecular ordering inside domains.
2.2.2
IR spectra probed with different polarizations
To get a deeper insight into molecular distribution and ordering in the investigated film, additional spectra with different infrared light polarizations (s and p) were collected. Generally, AFM-IR technique enables observation of different signal intensity from molecular vibrations depending on their orientation to laser polarization [48,49,47]. Namely, the signal is amplified when IR active bonds are oriented in the polarization, otherwise it weakens.
Comparison of selected representative AFM-IR spectra probed from different phases of sample with s and p polarization of infrared light was presented in Fig. 7
.
Generally, some differences between spectra from p- and s-polarization can be noticed. For example, band from hydrocarbon chains (at 1480 cm−1) weakens when s polarization is applied. This observation is in agreement with our previous results [48] and suggests that hydrocarbon chains (from phospholipids and F6H8) are predominantly oriented perpendicular to the gold surface. Further evidence for orientation of phospholipids (namely DPPC and DPPE) is partial decrease of absorption intensity at 1702 and 1744 cm−1 observed for s polarization. It can be explained with molecular modeling studies which revealed that DPPC ester groups vibrations are perpendicular to substrate surface, while in the case of DPPE they are oriented at 45°. Moreover, the intensity of the abovementioned band is much more noticeable in spectra probed from domains’ surrounding. This also confirms that phospholipids are in predominance distributed outside domains.
Additionally, s-polarized infrared activates numerous deformation vibrations in F6H8 molecule and as a result increased absorption below 1200 cm−1 appears. This band is characteristic to spectra collected from both phases (located inside and surrounding domains) which confirms that semifluorinated alkane is uniformly distributed on the surface.
Regarding spectral region attributed to amide I vibrations in SM and CsA in the spectra collected from domains a slight increase in intensity of band at 1665 cm−1 for s-polarized spectra is observed. It suggests that small quantity of SM is present inside domains. Larger differences can be noticed in the spectra probed from domains’ surrounding. For example, absorption in the range 1600-1650 cm−1 is increased for p-polarization. This observation may provide some information on the origin of this band. Namely, this peak is generated by amide vibrations (from SM and CsA) and in the case of SM can be strongly activated with the polarization direction. The increase of absorption intensity observed for p-polarized light suggests that SM molecules are more inclined in monolayer. This results are in agreement with previous studies by GIXD [50], which revealed that SM in other phospholipids environment adopts different orientation (more inclined) compared to films with cholesterol, where it is perpendicular to the surface.
Next, large intensity shift due to different light polarization may be observed regarding spectral bands in 1300-1450 cm−1 region, however, due to overlapping of bands from different compounds, this phenomenon does not provide information on molecular orientation.
2.2.3
PCA analysis
Multivariate data analysis here Principal Component Analysis (PCA) is commonly used for spectroscopic data sets in order to reduce large amount of acquired data and explore markers typical for groups of similar spectra. Herein, we have applied PCA to verify spectral variability across nano-metric features observable by AFM. Two PCA models were applied for spectra collected from TLL treated with CsA and F6H8 with s- and p-polarised laser light separately. Fig. 8
demonstrates score plot, which shows the separation of spectra, taken with p-polarised light, from the domains and from the neighbouring areas. Scores plot project each spectrum as a single point in the space of new variables called principal components.
This plot confirmed that spectra collected inside and outside domain are distinguishable based on functional groups oriented perpendicular to the surface. Moreover, the distribution of the spectra shows that spectra collected outside the domains are more similar to each other than those acquired inside the domains. This suggests that chemical structure and composition inside the domains is more heterogeneous than inside the surrounding phase. Corresponding loading plot shows spectral differences responsible for the clustering visible on the scores plot. The separation along PC-1, explaining 43% of total variance and it is determined by the band at 1480 cm−1 from CH2 stretching. Loading plot indicates that this band is more intense in the spectra acquired inside the domains (stars located on the positive side of PC-1). This suggests that hydrocarbon chains are more compactly packed inside the domains than outside which confirms presence of CsA (polypeptide with dense molecular structure). The separation along PC-1 is caused also by Amide II band from CsA and SM at 1540 cm−1, characteristic for spectra acquired inside the domains. Another very important factor, which determines the separation along PC-1 is a shift of band attributed to ν(C–F), ν(C–C), ρ(C–H), τ(C–H) in F6H8 from 1180 cm−1 (positive correlation with PC-1) to 1190 cm−1 (negative correlation with PC-1). This suggests that F6H8 molecule is in slightly different chemical environments inside and outside the domains (inside the domains due to interaction with CsA the vibration energy is different). Negative correlation of PC-1 is observed for Amide I vibrations (1630–1670 cm−1) and band from F6H8 (at 1264 cm−1), these bands are characteristic for all spectra collected outside the domains and some spectra acquired inside the domain. Separation along PC-2, which explains 24% of the total variance is also related with Amide II and CH2, CH3 deformational modes.
Second PCA model was applied to the spectra acquired with s-polarised laser light in order to get deeper insights into nanoscale distribution of chemical bonds oriented parallel to the surface, however no evident separation was observed (Fig. S.4 in Appendix A).
3
Conclusions
A variety of experiments conducted on biomimetic self-assembly allowed us to conclude on cyclosporine A and perfluorohexyloctane role in DES therapy. Langmuir monolayer technique complemented with BAM and AFM imaging demonstrated that F6H8 (regardless of the dose) does not influence interfacial properties, physical state and texture of DPPC/DPPE/SM mixed film. This confirms that semifluorinated alkane is a good supplement to the tear film lipid layer for use in the DES treatment. Furthermore, our studies revealed that immunosuppressant CsA expandinds DPPC/DPPE/SM monolayer and loosens molecular packing, and this can be reversed by the addition of F6H8. In this way, the fluidity of the artificial membrane remains unchanged. This is very important observation and proves that the combination of CsA and semifluorinated alkane can safely be applied in DES therapy. Additionally, we demonstrated that AFM-IR technique provided precise information about molecular distribution and orientation in the investigated biomimetic samples. F6H8 increases the availability of CsA, resulting in its more homogeneous distribution within the amphiphilic sublayer of the tear film (as proved by model studies), which is in consistence with previous hypotheses that F6H8 increases CsA dispersion [23]. We conclude that synthetic F6H8 is a suitable CsA carrier for the treatment of DES. Moreover, multicomponent Langmuir and Langmuir-Blodgett films investigated at ambient laboratory conditions provide a good model to study molecular ordering and packing in the field of drug delivery.
Appendices
Appendix A. Experimental section, Theoretical and experimental IR spectra, Supplementary figures (PDF)
Appendix B. Vibrations of ABA in CsA (GIF)
Appendix C. Vibrations of BMT in CsA (GIF)
Appendix D. Vibrations of MLE in CsA (GIF)
Appendix E. Vibrations of MVA in CsA (GIF)
Appendix F. Vibrations of VAL in CsA (GIF)
Declaration of Competing Interest
The authors declare no conflict of interest
Acknowledgments
The authors are grateful to Professor Wojciech M. Kwiatek (The Henryk Niewodniczański Institute of Nuclear Physics Polish Academy of Sciences) for providing measurement time on nanoIR2 instrument (purchased in frame of the project co-funded by the Małopolska Regional Operational Program Measure 5.1 Krakow Metropolitan Area as an important hub of the European Research Area for 2007-2013, project No. MRPO.05.01.00-12-013/15). This research was supported in part by PL-Grid Infrastructure.
Appendix A
Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110564.
Appendix A
Supplementary data
The following are Supplementary data to this article:
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COLSUB
110564
110564
S0927-7765(19)30708-8
10.1016/j.colsurfb.2019.110564
The Authors
Perfluorohexyloctane (F6H8) as a delivery agent for cyclosporine A in dry eye syndrome therapy – Langmuir monolayer study complemented with infrared nanospectroscopy
Anna
Chachaj-Brekiesz
a
*
chachaj@chemia.uj.edu.pl
Anita
Wnętrzak
a
Ewelina
Lipiec
b
c
Jan
Kobierski
d
Patrycja
Dynarowicz-Latka
a
a
Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland
Faculty of Chemistry
Jagiellonian University
Gronostajowa 2
Kraków
30-387
Poland
Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland
b
Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland
Faculty of Physics, Astronomy and Applied Computer Science
Jagiellonian University
Łojasiewicza 11
Kraków
30-348
Poland
Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland
c
The Henryk Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Kraków, Poland
The Henryk Niewodniczański Institute of Nuclear Physics
Polish Academy of Sciences
Radzikowskiego 152
Kraków
31-342
Poland
The Henryk Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Kraków, Poland
d
Department of Pharmaceutical Biophysics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland
Department of Pharmaceutical Biophysics
Faculty of Pharmacy
Jagiellonian University Medical College
Medyczna 9
Kraków
30-688
Poland
Department of Pharmaceutical Biophysics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland
⁎
Corresponding author.
Graphical abstract
Highlights
•
Mixed Langmuir monolayers were used to mimic polar lipids compartment of TTL.
•
CsA affects tear phospholipids assembly by loosening of molecular packing.
•
Effectiveness of F6H8 as delivery agent for CsA was confirmed.
•
Low-cost laboratory approach for membrane active drugs studies was proposed.
Abstract
One of the key challenges in dry eye syndrome therapy is to find a suitable carrier for immunosuppressant drug – cyclosporine A (CsA) – delivery to the eye. To investigate this issue, herein we present a methodology based on the combined analysis in macro- (Langmuir monolayers), micro- (Brewster angle microscopy) and nanoscale (atomic force microscopy and infrared nano-spectroscopy). The applied approach proves that CsA affects the phospholipid part of the tear film lipid layer by loosening molecular packing. This effect can be reversed by the addition of perfluorohexyloctane (F6H8). We have highlighted that F6H8 increases the availability of CsA and therefore is appropriate carrier for CsA topical delivery to the eye in the dry eye syndrome. In addition, the applied herein procedure provides a simple, low-cost laboratory tool for preliminary studies involving membrane active pharmaceuticals, preceding in vivo tests.
Abbreviations
AFM-IR
infrared nano-spectroscopy
ATR-FTIR
attenuated total reflection Fourier transform infrared spectroscopy
BAM
Brewster angle microscopy
CE
cholesteryl ester
CsA
cyclosporine A
DES
dry eye syndrome
DFT
density functional theory
DPPC
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DPPE
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine
F6H8
perfluorohexyloctane
LB
Langmuir-Blodgett
OAHFA
O-acyl-ω-hydroxyfatty acid
PCA
principal component analysis
SM
sphingomyelin
TLL
tear film lipid layer
WE
wax ester
Keywords
LB films
Tear film
Cyclosporine A
Semifluorinated alkanes
AFM-IR
KBJ00000000011934
2019-12-21T02:10:04
S300.2
S300
S0927-7765(19)30708-8
10.1016/j.colsurfb.2019.110564
COLSUB
0927-7765
110564
110564
FLA
NON-CRC
UNLIMITED
ICM
2019-10-07T13:50:20Z
09277765/v184sC/S0927776519307088/main.xml
125338
MAIN
JA 5.6.0 ARTICLE
FULL-TEXT
09277765/v184sC/S0927776519307088/main.assets/ga1.sml
12306
IMAGE-THUMBNAIL
09277765/v184sC/S0927776519307088/main.assets/gr1.sml
3812
IMAGE-THUMBNAIL
09277765/v184sC/S0927776519307088/main.assets/gr2.sml
13415
IMAGE-THUMBNAIL
09277765/v184sC/S0927776519307088/main.assets/gr3.sml
5407
IMAGE-THUMBNAIL
09277765/v184sC/S0927776519307088/main.assets/gr4.sml
4944
IMAGE-THUMBNAIL
09277765/v184sC/S0927776519307088/main.assets/gr5.sml
8631
IMAGE-THUMBNAIL
09277765/v184sC/S0927776519307088/main.assets/gr6.sml
14596
IMAGE-THUMBNAIL
09277765/v184sC/S0927776519307088/main.assets/gr7.sml
8379
IMAGE-THUMBNAIL
09277765/v184sC/S0927776519307088/main.assets/gr8.sml
7757
IMAGE-THUMBNAIL
09277765/v184sC/S0927776519307088/main.assets/ga1.jpg
20053
IMAGE-DOWNSAMPLED
09277765/v184sC/S0927776519307088/main.assets/gr1.jpg
28401
IMAGE-DOWNSAMPLED
09277765/v184sC/S0927776519307088/main.assets/gr2.jpg
51888
IMAGE-DOWNSAMPLED
09277765/v184sC/S0927776519307088/main.assets/gr3.jpg
32770
IMAGE-DOWNSAMPLED
09277765/v184sC/S0927776519307088/main.assets/gr4.jpg
23277
IMAGE-DOWNSAMPLED
09277765/v184sC/S0927776519307088/main.assets/gr5.jpg
44833
IMAGE-DOWNSAMPLED
09277765/v184sC/S0927776519307088/main.assets/gr6.jpg
54451
IMAGE-DOWNSAMPLED
09277765/v184sC/S0927776519307088/main.assets/gr7.jpg
36367
IMAGE-DOWNSAMPLED
09277765/v184sC/S0927776519307088/main.assets/gr8.jpg
52420
IMAGE-DOWNSAMPLED
09277765/v184sC/S0927776519307088/main.assets/mmc2.gif
1551673
VIDEO
09277765/v184sC/S0927776519307088/main.assets/mmc3.gif
1547074
VIDEO
09277765/v184sC/S0927776519307088/main.assets/mmc4.gif
1564519
VIDEO
09277765/v184sC/S0927776519307088/main.assets/mmc5.gif
1560492
VIDEO
09277765/v184sC/S0927776519307088/main.assets/mmc6.gif
1567152
VIDEO
09277765/v184sC/S0927776519307088/main.assets/mmc1.pdf
943939
APPLICATION
09277765/v184sC/S0927776519307088/main.pdf
1854477
MAIN
1.7 6.5
DISTILLED OPTIMIZED BOOKMARKED
09277765/v184sC/S0927776519307088/main.raw
56844
S0927-7765(19)X0009-0
COLSUB
0927-7765
184
C
20191201
S0927-7765(19)30708-8
10.1016/j.colsurfb.2019.110564
main.pdf
PDF
1.7
local
1577101671680
collectiebehoudsniveau 1
2019-12-23T11:47:15.407+01:00
local
1577101672192
0
SHA-512
438102e9c380af6d366605477890572e0f08ab51698c80ac22fbb89d15675e394da3f30daa5b90209eabffb5f57d9b5e59e39bc05f042b708d526c357816c53b
java.security.MessageDigest
1854477
Adobe Acrobat Document
1.7
DIAS
62
DIAS tentative identification
main.pdf
local
1577101672193
0
SHA-512
200b428921582fcc5b45a265625e8d4dd06c2bcef8a313d2dcddd5a264ef4365b22b00d348b46e25388387327cd5ded8eaede44cf5acfbb4b6f8726bdc96f4a3
java.security.MessageDigest
56844
not checked
main.raw
local
1577101672194
0
SHA-512
706ab80660323a9befec56add48f0d2fc69a87f08b567ed4e7d34c93a1e8cfa1dfaa9154ae5dd26ff22fde5bcc03434b4a3ee06ca6913dcfe01c1060125fbd3f
java.security.MessageDigest
125338
not checked
main.xml
local
1577101672195
0
SHA-512
2d200ea7d9a7e398269f34a1d5230ffa7e7f5b342d2492312201dd969b215eb9c410cd1827f75e431ccfde75c272a1f314b839318cd0ccf8f3aa464849b0e3d0
java.security.MessageDigest
6570
not checked
si1.svg
local
1577101672196
0
SHA-512
330fa5160b646dfeb51d1cd7027c7fa84d8464bfae493a128d10ba2ed75b004eace92c8cccedabb790d76eaa52649cf70f018440ab8111872d5fad5dfe375686
java.security.MessageDigest
19244
not checked
si2.svg
local
1577101672197
0
SHA-512
bae82041a48778320447ad030fa52aa8898710df9623a1b485fd0d2151d82cd5710e4044e5809326ff23c077287279fea8c7b8d1c779dea6ee45c672bdc1519a
java.security.MessageDigest
20053
not checked
ga1.jpg
local
1577101672198
0
SHA-512
c101e4df03da5c5192c6931799f21c3ff93c1d90767da7109e5f7907ca0fd5d6bc875101ceb1ad8fb6ed3c27e46166d089889705aa6cfcfd493c15cecc4f9565
java.security.MessageDigest
12306
not checked
ga1.sml
local
1577101672199
0
SHA-512
1629eb74e558697940de2ee9c82ed125ab582f039ed7a3348a3afb7e1a5372e93ba18fc50dc21c36b59665ecf96f46c7256be7486b20c128d7939471f127e4ec
java.security.MessageDigest
28401
not checked
gr1.jpg
local
1577101672200
0
SHA-512
49de305f9db2c78e574b5438cfda433e2cd917aac425664139b6e6dc586d2ac1e30b6894007fb878be2643fe982ec9c4e929b40b44904aa5df2cc8e7fb6ec0bf
java.security.MessageDigest
3812
not checked
gr1.sml
local
1577101672201
0
SHA-512
788bb40d278195449c0c17e543fdb68836e365247f08c4858fe5e3b948e5939da54c3ab9b90a7dc84d97ed96e68957c307fa0cad50f8507c4f313c7e5d005876
java.security.MessageDigest
51888
not checked
gr2.jpg
local
1577101672202
0
SHA-512
630ce9af1ec1673a208a41ec1ddbbfc418264ae404bf344d5e77d7142c284d01ae45da221a7fcd1344c04781e932d572879f408f903f08b8492c5edd99ba36d0
java.security.MessageDigest
13415
not checked
gr2.sml
local
1577101672203
0
SHA-512
71c8eca3345875ee5bf0c4794c0e4255e9fd6d4a7877a103168a70b1d2a07ecfd1fe0d8d604517fb91d5e960790c53123283ebb4f589440f99e35632b5d3d453
java.security.MessageDigest
32770
not checked
gr3.jpg
local
1577101672204
0
SHA-512
03c66e1a0c0c3d7d34bcdbb57209d8cc4bddba187c034bfc75be5717cc9a410b555dd22dc60e34f8b5ab8947ef4e6b250def30478faa2bb960febf08ad0bec50
java.security.MessageDigest
5407
not checked
gr3.sml
local
1577101672205
0
SHA-512
d824822dafb3c40991fb3e08e1b0834e25fddbc7190bc06e936f7075454cf9dba962605bdf81cc429e632586aaf0ed3f5bea9857142d8779c20f723f920f6dca
java.security.MessageDigest
23277
not checked
gr4.jpg
local
1577101672206
0
SHA-512
ef0e5ce992291e9b761b20a05b7d7a9fa50656b7c92986fd1e7fa617e3fd25a687b93c7d362270f84f73f6c58fd973aadb314f1f35a346f0cfe6543d910c493f
java.security.MessageDigest
4944
not checked
gr4.sml
local
1577101672207
0
SHA-512
e02b8a080701865ba0f4cbd5d509c4afa8889466779596b24f9bb88317db29c0143346fc43dfbbdc1355baa0df3d94cf3f16ce1d52da83ef51644334cda2eb92
java.security.MessageDigest
44833
not checked
gr5.jpg
local
1577101672208
0
SHA-512
f66ad17a09b494c69040882cc5c6ab4ae4ffb3b2c88cbe525770c9d0b7064413ebd406d5c3e1267b14ae537bea4333a6877180e357621e980750070a0e707445
java.security.MessageDigest
8631
not checked
gr5.sml
local
1577101672209
0
SHA-512
d1cb6c1e69a3454343c1aca0a7a3ccb6271f92543d56490175b8eca0a729067ee2edbea40bf1e75bad2820f6324e003432ed0b902fef79be09d11e868045b5d8
java.security.MessageDigest
54451
not checked
gr6.jpg
local
1577101672210
0
SHA-512
016f27eaffffb06eb3d9574fb992f27095a5629e5ae6801f0a13512d3bae71794d77d01a0d13d648145daa6f43e4432adb9b851c4980362179b123bb6dfa7f2b
java.security.MessageDigest
14596
not checked
gr6.sml
local
1577101672211
0
SHA-512
7eb5e3f0692db651235de85b72a714063db05029699b351addbfa492229c450d5befe07b8970fc1101be72c079983bfa47d6d3de1faf1285a4df91b04338752b
java.security.MessageDigest
36367
not checked
gr7.jpg
local
1577101672212
0
SHA-512
18ae09632197a504ed06982bcdc4aa149ce48bc810aea4ae25a1363b847b6ba99c3ec8c54d9d5a6f19f837836046a3829454f129b052d91be4f37e3803cc9b3c
java.security.MessageDigest
8379
not checked
gr7.sml
local
1577101672213
0
SHA-512
ca97fc1d0bbe98915a0a4b0248605737c455731491f4d4563352d659ab2a00f2c57a47ea66cb4aacb0ff1f2127601cc50539261bcec8c09885a11ddb046c1890
java.security.MessageDigest
52420
not checked
gr8.jpg
local
1577101672214
0
SHA-512
a2989ac5cce68d9fcc514eeee9205ac322bcf5d82be179af6d0de0980ee6db402377055683fea0e7a40b486815a6ed59de0978bc2661af6b6c3e36b4a4c521f3
java.security.MessageDigest
7757
not checked
gr8.sml
local
1577101672215
0
SHA-512
95541a8ca0132285f6f915a7a435ee782788216ff4e61c583cf61eb30770a373d01f66bf241c1151cb43a635e45c2c0d86c505a9233544e669a6177656706c6e
java.security.MessageDigest
943939
not checked
mmc1.pdf
local
1577101672216
0
SHA-512
a83efd9b3fa8c81b99a0e82d60ad107e792688cdff7841dd3e69ae51eb9353ea4a95e7dbd2a9ea88d4c67772fffa87a9e48b9ba8e8109438385e5026d0f7fe71
java.security.MessageDigest
1551673
not checked
mmc2.gif
local
1577101672217
0
SHA-512
3efa46868584f3c51462582a8a8b54d6ae1ea7cf2a27460852ac99b8e49171e8c825dbdbafd57a9a317dc32332639725a377c2d66d62c1c0cf1c103d5b55b9b5
java.security.MessageDigest
1547074
not checked
mmc3.gif
local
1577101672218
0
SHA-512
158c06ca30e0773226abf4ef548947cc65679a3a5a857dc3a8b0b1d7d7f78ac9553c84e600dbe1a3b8658c7ff623af1cc6306d71c46abad19df2fca5e3a6ce52
java.security.MessageDigest
1564519
not checked
mmc4.gif
local
1577101672219
0
SHA-512
93ef295ce0010d52d1318862618d39539c8a6f51ed6792cdabd6dedf4c6d94561f5d20ffc4b2a4601582768a344847777f7dad3d3c759351a5560800087f1576
java.security.MessageDigest
1560492
not checked
mmc5.gif
local
1577101672220
0
SHA-512
3decc698cef1a70dd9af54af1a839e3954ca85a1bd27f6ecb372559b9be2547b86ecfc9acb1bf67ca0cea6d33bfae6f6f9a90872590de83f8250b7ca1c0ae912
java.security.MessageDigest
1567152
not checked
mmc6.gif
local
1577101672221
0
SHA-512
b59c04c31dfd3554d3e389a95ef619ddce1c5990dc4255f848d33ad989bbaf24a59178b220479b5e4052b35262e0b9c5f57e1715498d2060ca49df6a67e3b73d
java.security.MessageDigest
24502
not checked
metadata.xml
free
00001
The Authors
KB-agent-id
1
supplier
KB-owner-id
00001
KB-agent-id
1
Elsevier
organization
The Authors
ingestion
2019-12-23T11:47:15.407+01:00
Connector
software
Digitaal Magazijn release 1.5
ejournals_esp_1
streamprofile
ingestion2019-12-23T13:18:19.740+01:00Generic IngestsoftwareDigitaal Magazijn release 1.5