3.1. Liposomes’ Physicochemical Characterization
presents the average size, PDI, and zeta potential of the produced QT-LLs. Regarding the results of average size, it can be noted that QT-LL 0 (control) presents statistically lower values (p
< 0.05) when compared with those containing quercetin concentrations above 50 µg/mL, while for QT-LL 25 no difference (p
> 0.05) was observed. In addition, increasing quercetin concentrations above 100 µg/mL led to higher values of average size. The results related to PDI showed that all samples presented a low PDI (≤0.29) even considering their significant differences. All QT-LLs presented zeta potential values around −45 mV with no statistical differences; regarding these values, it is possible to say that the encapsulation of quercetin does not influence the surface of the liposomes. LLs 0, 25, 50, and 75 were homogeneous; however, QT-LLs 100, 125, and 150 showed signs of instability (precipitation): QT-LL 100 and 125 after 72 h, and QT-LL 150 after 48 h of observation (Figure 1
It is known that smaller particles present a higher exposed surface area which can lead to a faster release of encapsulated drugs; however, smaller particles also have an increased risk of aggregation during storage. In view of this, it is important to produce liposomes with adequate size (<200 nm) and low values of PDI, thus reaching maximum stability by a good control of particles’ size. It is also important to mention that the reproducibility of parameters such as stability and release is directly connected to a low PDI (≤0.4), since a high PDI means that there is no uniformity in the size distribution of the sample [21
]. In relation to the zeta potential parameter, results around ± 45 mV of surface charge can positively influence its stability through electrostatic repulsion among the liposomes. In the present study, the negative charge demonstrated by zeta potential could be associated to the phosphate groups of the mixture of phospholipids composing the lecithin (Lipoid S45).
This behavior was also reported by Gibis, Vogt, and Weiss [26
] for 1% (w
) lecithin liposomes, which had negative surface charges regardless of the exposure to different pH values; the magnitude of surface charges decreased from −21.8 at pH 3 to −57.7 at pH 11. More recently, Talón et al. [21
] also reported a zeta potential value of −45.7 mV for liposomes based on lecithin and maldodextrin. This charge was inverted when chitosan was incorporated into the system, leading to a zeta potential of +61.5 mV. Our zeta potential results were not influenced by the amount of quercetin loaded in lecithin liposomes, However, a different pattern was observed for average size values; increasing the concentration of quercetin resulted in decreased stability of QT-LLs.
3.5. Film Thickness, Moisture Content (MC), Solubility (S %), Water Vapor Permeability (WVP), and Contact Angle (CA)
The moisture-binding abilities of films can affect significantly their physical and barrier properties; thus knowledge of the content and affinity of the film matrix to water is a key parameter when choosing a film for specific applications. Table 4
shows the values of thickness, moisture content (MC), solubility (S %), and water vapor permeability (WVP) of the control film and the films with QT-LL 75 immobilized in different concentrations (B–F).
The incorporation of different concentrations of QT-LL 75 in galactomannan-based films did not lead to statistical differences (p
> 0.05) for thickness, MC, and S %; in addition, these values were similar to those reported by [3
] working with similar galactomannan-based films. Other authors have reported values of thickness and discussed its importance in the characterization of galactomannan films or blends containing galactomannans. For example, films based on D. regia
galactomannan with different molecular weight and interaction with k-carrageenan presented values of thickness between 37 and 59 µm [31
], while the galactomannan extracted from G. triacanthos
and its combination with soy protein isolates showed thickness of 76 ± 8 and 106 ± 3 µm, respectively [42
]. Therefore, variations in thickness values could be observed when molecular interactions occur between the components of the blend, and also caused by acid solvents used in the preparation of the components [45
Modifications in the MC can affect the physicochemical and thermomechanical properties of the films; the addition of glycerol is one of the main modifiers, which is explained by its hydrophilic nature and the attraction of water into the polymer matrix, thus creating more mobile regions with greater inter-chain distances in the polymer matrix [44
]. Our results showed that the films’ MC were around 22% and the addition of QT-LL 75 did not influence this parameter; however, a different behavior was observed for the same galactomannan film incorporating 0.2% of lactoferrin (15.67 ± 5.51%) [3
]. According to the authors, the decreased MC values could be associated with the fact that lactoferrin presents both hydrophilic and hydrophobic parts in its conformation; the higher concentration of lactoferrin into the film may reduce the availability of hydroxyl groups to interact with water, resulting in decreased MC values. Tara gum (TG) films and nanocomposites, in this case chitosan nanoparticles incorporated in TG films, also presented lower values of MC, respectively, 14 and 13%. The authors explained that the nanocomposites presented a more compact matrix, which allowed them to occupy more free volume into the polymeric structure, thus reducing MC values [44
]. Other works reported MC values similar to the ones of this work; for example, 31.01 ± 0.80% for chitosan-based films, 21–7% for chitosan films incorporated with different amounts of black soybean seed coat extract [47
], and 14–7% for chitosan films neutralized with citrate and acetate buffers, added or not with glycerol [45
The values of the films’ solubility were approximately 70% and this was not influenced by the addition of QT-LL 75 in different concentrations. This great solubility behavior was also observed for films produced with D. regia
galactomannan with different molecular weights (between 69 and 76%) [31
]. In this same work, the galactomannan was blended with k-carrageenan and all of the obtained films presented 100% solubility, which was considered to be a result of the k-carrageenan sulphate functional groups which can form hydrogen bonds with water molecules helping to disrupt the film and increasing its solubility. The solubility of Tara gum films was 24% and different when compared to our results; and the addition of chitosan and chitosan nanoparticles (both at 15% w/w content) significantly decreased the films’ solubility (%) to 10.37 ± 2.31 and 6.16 ± 1.17, respectively [44
]. The quercetin-loaded lecithin liposomes used in this work have average sizes ranging from 93 to 131 nm, different from the chitosan nanostructures used in the above-mentioned work (180.25 nm). The average size, in addition to the strong interaction between chitosan nanoparticles and Tara gum chains, may have influenced the Tara gum film structure, justifying the differences obtained for S % when compared to our galactomannan-based films.
WVP values ranged from 7.27 ± 0.80 (A) to 11.41 ± 1.05 × 10−7
(D) g/(h·m·Pa). The presence of QT-LL 75 at 0.2% (C), 0.3% (D), and 0.5% (F) significantly increased (p
< 0.05) the WVP results, leading to values up to 1.9-fold higher than the galactomannan-based film without liposome (A). On the other hand, the WVP values of B and E films were similar to the control film. According to Antoniou et al. [48
], the hydrophilic pattern and the water attracted into the film contribute to create regions of higher water mobility, with greater inter-chain distances in the galactomannan film. In view of this, it is possible to consider that these WVP results are in agreement with those of MC and S, confirming the hydrophilicity of all of the films. Indeed, the differences in WVP values of films C, D, and F can be associated with the irregular surface of the film, as reported by SEM results (Section 3.4.1
). Since the liposomes are immobilized on the surface of the films, the free spaces release a passage of water vapor through the chains of the galactomannan; thus, we hypothesize that the increase in WVP values occurs due to a lower compression of the film matrix, resulting in a modified diffusivity.
The water vapor pattern demonstrated by our films was also reported by other authors working with galactomannans; for instance, WVP values for films produced with the C. grandis
galactomannan and different bioactive molecules ranged from 5.60 ± 0.39 to 22.43 ± 0.21 × 10−7
], while those for films produced with D. regia
galactomannan with different molecular weights varied between 7.55 and 12.70 × 10−11
]. Easy water-vapor permeation was demonstrated for Tara gum films and associated with the high glycerol concentration used in the film-forming solution [44
]; however, when chitosan and chitosan nanostructures were added to the galactomannan-based films, they decreased WVP values in a different manner than what was demonstrated for our films with incorporated quercetin-loaded lecithin liposomes. According to the authors of the above-mentioned study, the chitosan nanoparticles have a greater ability to occupy the empty spaces of the porous film matrix, thus creating a more compact structure in the films and impairing the permeation of water, which is associated with decreased WVP results. Similar decreases in WVP values were reported when chitosan film incorporated different amounts of black soybean seed coat extract [47
], carvacrol extract [49
], and apple polyphenol [50
]; in this case, reduced WVP values could be related to the compact and dense networks formed through intermolecular interactions between the immobilized molecules and chitosan chains.
The determination of the contact angle (CA) is a simple way to evaluate the degree of hydrophobicity of a material layered on a surface or immobilized on a ready surface. Commonly, CA values lower than 90° are related to hydrophilic materials [3
]. CA for all of the films (A–F) was measured at 5 s and additionally 30 s in order to understand the pattern of hydrophobicity of the films over time. The results demonstrate that all of the films were essentially hydrophobic with no statistical differences between them, including when analyzed over time (Table 5
). The distribution of QT-LL 75 on the surface of the films was quite homogeneous and did not statistically influence CA values, as confirmed by S% and MC results (Table 4
). The C. grandis
galactomannan film reported by Albuquerque et al. [3
] presented a lower CA, being considered more hydrophilic than any of the films in this study. Considering that the same source of galactomannan was used in both studies, it is possible to predict that different procedure conditions, the period of study, and environmental conditions could have influenced the final films. However, as mentioned by the authors, this difference can be useful for specific applications, since the relatively hydrophobic pattern of the film allows the graduated delivery of immobilized compounds by helping them to remain in the film until the complete solubilization of the matrix.
Other works evaluated the hydrophobic character of films produced by polymers from different sources. For example, nanofibers obtained from low-cost agro-industrial by-products (soybean hulls and pods) were used as reinforcers of soy protein films. The authors performed an extensive characterization and reported a reduction in the hydrophilicity of the reinforced films’ surface, as revealed by higher CA values; they also suggested that it occurred due to the interactions between components and attributed this effect to the properties of the nanomaterials [29
]. The film-forming properties of the galactomannan fraction extracted from G. triacanthos
and its ability to improve the properties of soy protein films were also evaluated [42
]. The results revealed that the galactomannan film had a more hydrophobic surface when compared to the pure soy protein film; however, a synergic effect was achieved by the combination of both components, probably derived from their interactions. In addition, the correlation of WVP with the contact angle was inferred, and they reported that the more hydrophobic surface led to a smaller WVP, as noted in our work.
Considering the above-mentioned results, it is possible to state that quercetin has been successfully encapsulated in lecithin liposomes by a simple technique were we used a low concentration of lecithin (Lipoid S45 at 25 mg/mL), which was injected, drop by drop, during only 2 min, in 23 mL of distilled water under magnetic stirring (600 rpm). This suspension was left in magnetic stirring for a further 8 min and then submitted to a vigorous shaking on a simple mixer for 2 min at room temperature. Other references also used ethanol injection and similar phospholipids; however, significant differences in technique could be seen:
Higher concentration of phospholipids, for example, the 60 mg/mL of Lecinova®
or Lipoid S75 used for the development of liposomes by Gil et al. [18
Other reagents and sophisticated equipment, as mentioned by AbouSamra, Elgohary, and Mansy [19
] on the development of pirfenidone-loaded lecithin core nanocapsules, which included Span 60, Tween 80, and chloroform, in addition to a homogenizer that reached more than 20,000 rpm;
Longer experimental period, for example the stirring overnight of the suspensions containing whey lecithin, maltodextrin, eugenol, oleic acid, and chitosan; these suspensions also required a high-pressure homogenizer for complete homogenization [21