3.1. Lipid Nanoparticles Characterization
As reported in the literature [29
], the use of solid lipids to form the lipid nanocarrier core leads to nanoparticles defined as SLNs while the use of mixtures of solid and liquid lipids results in NLCs. Therefore, in this work, lipid nanoparticles loading rosemary EO were regarded as NLCs, being that their lipid phase was a mixture of solid lipid (cetyl palmitate) and liquid lipid (rosemary EO).
As shown in Table 3
, unloaded lipid nanoparticles containing only the solid lipid cetyl palmitate (SLN A) showed small sizes (36.45 nm) and narrow dimensional distribution (polydispersity index 0.208), in accordance with previous studies [27
]. When rosemary oil was mixed in different ratios with cetyl palmitate, NLCs with different mean sizes and polydispersity indexes were obtained. When the total amount of lipid phase (solid lipid plus oil) was maintained constant (7% w
, NLC BEO1–4), an increment of oil content led to an increase of mean particle size. For these lipid nanoparticles, polydispersity index values were higher than 0.300, thus indicating that the distribution did not consist of a single size mode.
As illustrated in Figure 1
, two different populations of lipid nanoparticles were observed in NLCs BEO1–4, one with small sizes (mean size 22 nm) and the other with bigger sizes (mean size 312 nm). At a lower rosemary EO content (1–2% w
), the percentage of small sized nanoparticles was higher than that of large nanoparticles. When the amount of essential oil used to prepare these nanocarriers was raised to 4% w
, the percentage of large nanoparticles became preponderant.
The opposite trend was observed for lipid nanoparticles obtained using a constant amount of solid lipid (7% w
) while increasing the content of rosemary EO (1–3% w
). These NLCs (CEO1–3) showed a decrease of particle sizes and polydispersity index values as the percentage of rosemary EO increased (see Table 3
). NLC CEO1 and NLC CEO2, loading 1–2% of rosemary EO respectively, showed two populations of nanoparticles, having small (30 nm) and large (235 nm) mean size, while NLC CEO3 (rosemary EO 3%) contained a single population of small nanoparticles (Figure 2
These results point out that the ratio solid lipid/liquid lipid plays a key role in determining NLC size and dimensional distribution, as already reported in literature [31
]. The interactions occurring among NLC components may change depending on the solid lipid/liquid lipid ratio. These interactions may affect the nanoparticle curvature radius, thus leading to nanoparticles with different mean sizes.
Morphological analyses were performed by transmission electron microscopy (TEM) only on samples showing a single population of nanoparticles. TEM imaging of SLN A and NLC CEO3 showed that nanoparticles were round in shape (Figure 3
). No evident difference could be observed between SLN A and NLC CEO3 TEM images as TEM provides information only about the morphology and the external surface of the nanoparticles.
While the content of rosemary EO strongly affected the mean particle size and dimensional distribution, zeta potential values were similar for all the nanoparticles under investigation. It is interesting to note that all rosemary EO-loaded nanoparticles showed zeta potential similar to that of unloaded SLNs, thus suggesting that the incorporation of rosemary EO into these nanoparticles did not influence their superficial charge.
The data reported in Table 3
highlight that the higher the rosemary EO content, the lower the PIT values, for both series of NLCs. As shown in Figure 4
, a linear relationship between percentages of rosemary EO used to prepare the nanocarriers and PIT values of the corresponding nanoparticles was observed (r2
= 0.985 for NLC BEO1–4; r2
= 0.983 for NLC CEO1–3).
Concentration-dependent interactions occurring between rosemary EO and lipid nanoparticles components could explain PIT lowering as rosemary EO content increased. As reported in the literature [32
], rosemary EO hydrophilic lipophilic balance (HLB) is 16.5 while cetyl palmitate has an HLB value of 10.0. Therefore, increasing the percentage of rosemary EO in the lipid phase would lead to an increase in hydrophilicity of the lipid mixture. In conventional emulsions, an increase in hydrophilicity is expected to lead to an increase in PIT values [33
]. As NLC BEO1–4 and CEO1–3 contain both solid and liquid lipids, they could be classified as multiple NLCs [17
]. Being comprised of complex structures, with a matrix consisting of oil droplets embedded in solid lipids and a shell of surfactants, the interactions among different components could affect PIT values differently from conventional emulsions. Therefore, further studies have been planned to elucidate the interactions occurring between rosemary EO and other nanoparticle constituents.
As only NLC CEO3 showed a single population of nanoparticles, we selected this colloidal dispersion for stability tests. No significant changes of particles size and polydispersity indexes were observed during storage at room temperature for two months (data not shown). Therefore, we used NLC CEO3 to prepare the gel vehicles for in vivo topical application.
All gels showed no change in their organoleptic properties during storage in non-transparent containers for one month at room temperature.
3.2. In Vivo Evaluation of Gel Formulations
The ability of SLNs and NLCs to increase drug skin permeation has been attributed to different factors such as occlusive properties, specific drug-carrier interactions with the skin and close contact with the skin’s outermost layers due to their small size [34
]. After topical application, SLNs and NLCs form a continuous and dense film on the cutaneous surface that prevents or decreases water loss from the skin, thus increasing skin hydration and, hence, drug skin permeation [37
]. Wissing and Müller [38
] performed an investigation on human volunteers, studying the hydrating and viscoelasticity effects of an O/W cream containing SLNs in comparison with the same vehicle without SLNs. The results of this study showed that the cream containing SLNs provided a higher increase in skin hydration than the conventional formulation, while skin elasticity was not significantly affected after a 28-day treatment with these formulations, likely owing to the young age of the volunteers enrolled in the study.
Therefore, in this work, the effects of rosemary EO-loaded lipid nanoparticles on skin hydration and elasticity from gel vehicles was assessed using a gel containing unloaded SLNs as a control.
We chose gel formulations as a vehicle for rosemary EO-loaded NLCs in order to avoid the interactions between vehicle components and NLCs, which may occur in complex vehicles such as emulsions and could interfere with the ability of NLCs to affect skin hydration and elasticity.
The results of in vivo tests on skin hydration were expressed as hydration difference (Δ hydration) between baseline values and values recorded after a one-week treatment with the gels under investigation. As shown in Figure 5
, topical treatment with gel A, which did not contain the active ingredient or lipid nanoparticles, did not lead to any change in skin hydration. Gels B and B1, incorporating 1.5% and 3.0% w
free rosemary EO, respectively, enhanced skin hydration but no significant difference (p
> 0.05) was observed between Gel B and B1, thus suggesting that an increase in rosemary EO concentration in the vehicle was not effective in further improving skin hydration. Topical application of gel C, containing only unloaded SLN, resulted in a hydration improvement similar to that obtained from gel B and B1, thus confirming the ability of SLNs to act as a skin-hydrating factor. After a one-week treatment, rosemary EO-loaded NLCs in gel vehicles (gels D and D1) provided Δ hydration values greater than those observed for gels B, B1 and C, but no significant difference (p
> 0.05) was observed between gels containing different percentages of rosemary EO-loaded NLC. These results highlight the usefulness of loading rosemary EO into lipid nanoparticles, supporting the findings of previous studies on rosemary extracts that reported an increase in activity upon loading these extracts into nanoparticles [39
shows that skin treatment with gels A, B and B1 did not alter elasticity values, while all the gels containing lipid nanoparticles provided a slight but significant increase in skin elasticity (p
< 0.05 for all comparisons between gels containing lipid nanoparticles and gels without nanoparticles). However, it is interesting to note the all subjects showed high elasticity values before the treatment with the gels under investigation. Therefore, only moderate increases of this parameter could be expected. Analogously to skin hydration data, no significant difference was observed between elasticity values obtained from gels D and D1 (p
< 0.05). In addition, our data showed a moderate effect on skin elasticity of the gel containing unloaded SLNs (gel C), which contrasts with the results reported by Wissing and Müller [38
], who did not find any change in this parameter upon topical application of SLNs. The effect of unloaded SLNs on skin elasticity observed in our work could be attributed to the older age of volunteers enrolled in this investigation. However, gel C provided a significantly lower increase in skin elasticity than gels D and D1 (p
< 0.05 for both comparisons), thus proving better activity from lipid nanoparticles loading rosemary essential oil.
In conclusion, the results of this study pointed out that in vivo topical application of rosemary essential oil from gel vehicles showed a hydrating effect that remarkably increased upon rosemary EO loading into lipid nanoparticles. A slight but significant effect on skin elasticity could be detected only after topical application of gel vehicles containing unloaded SLNs or rosemary EO-loaded NLCs. Therefore, loading rosemary essential oil into lipid nanoparticles seems to be a promising strategy to improve its topical benefits and for designing topical formulations for the treatment of cutaneous alterations involving loss of skin hydration and elasticity.