Preparation of the Formulations
The solubility of LE in different lipids is given in Table II. LE showed high solubility in Compritol® ATO 888 (0.11207 mg/mL), followed by cetyl stearyl alcohol (0.08084 mg/mL), Compritol® E ATO (0.04144 mg/mL) and 1-tetradecanol (0.04009 mg/mL). Compritol® ATO 888 is a blend of two different esters of behenic acid with glycerol (37). Cetyl stearyl alcohol is a mixed lipid consisting of the combination of long carbon rings of cetyl and stearyl alcohol. This lipid allows the polar molecules present in surfactants to form a bilayer structure with the outer surface molecules. This bilayer structure can provide a higher rate of drug entrapping capacity by reducing the possibility of lipid crystallization. In addition to this advantage, since the molecules in the polar part of cetyl stearyl alcohol cause a high rate of water retention in their outer structure, a tendency to gel can also be seen. (38). In classical emulsions prepared after lipid imaging study, a fluid formulation without phase separation in Compritol® ATO 888 and cetyl stearyl alcohol was obtained. Shimojo et al., in which NLC loaded with resveratrol were formulated, Compritol® ATO 888 as solid lipid, Miglyol® 812 as liquid lipid, and poloxamer® 188 as a surfactant were used in varying proportions. They reported that there was no phase separation in the classical emulsions they prepared after the solubility studies (39). In another study, compritol was used to obtain dexamethasone-loaded SLN particles because it dissolves the active substance at a high rate (0.08388 mg/ mL) and forms a fluid emulsion (40). In a study by Vieira et al., NLC formulations prepared which containing cetyl stearyl alcohol with various lipids (Imwitor® 900K, Kolliwax® GMS II and Dynasan® 116) as solid lipids and sucupira oil as oil. It has been reported that formulations prepared using cetyl stearyl alcohol are more fluid and maintain their homogeneity (41). The uniformity and homogenicity of SLN and NLC prepared using cetyl stearyl alcohol and Compritol® 888 ATO were also attributed to the efficiency of the emulsion step.
Table II Solubility of LE in different lipids, oils, and surfactants
Solid Lipids
|
Solubility of the drug (mg/mL)
|
1-tetradecanol
|
0.04009c ±0.0011
|
Cetyl alcohol
|
0.04322c ±0.0014
|
Cetyl palmitate
|
0.04811c±0.0017
|
Cetyl stearyl alcohol
|
0.08084b±0.0019
|
Compritol® E ATO
|
0.04144c±0.0017
|
Compritol® 888 ATO
|
0.11207a±0.0022
|
Cutina® CP
|
0.04716c±0.0010
|
Geleol™
|
0.04763c±0.0013
|
Gelucire® 50/13
|
0.04311c±0.0024
|
Imwitor® 491
|
0.04877c±0.0020
|
Imwitor® 900K
|
0.04664c±0.0014
|
Nacol® 16-98
|
0.04407c±0.0017
|
Softisan® 141
|
0.04729c±0.0023
|
Softisan® 601
|
0.04308c±0.0018
|
Stearic acid
|
0.05114c±0.0021
|
Oils
|
|
Castor oil
|
0.05812b±0.0015
|
Isopropyl myristate
|
0.06899b±0.0022
|
Oleic acid
|
0.09977a±0.0012
|
Olive oil
|
0.05707b±0.0017
|
Surfactants
|
|
|
0.09105b±0.0014
|
|
0.09983b±0.0019
|
|
0.12039b±0.0017
|
Lutrol® F68
|
0.16328a±0.0020
|
Tween® 80
|
0.10116b±0.0016
|
(a-c): Statistically different between the other component (p≤0.05) |
LE showed high solubility in oleic acid (0.09977 mg/mL), followed by isopropyl myristate (0.06899 mg/mL), castor oil (0.05812 mg/mL) and olive oil (0.05707 mg/mL). While formulations prepared using oleic acid were stable at room temperature, it was observed that phase separation and precipitation occurred after a while in formulations prepared using other oils, which supports the effect of oleic acid in the formation of a homogeneous colloidal system. Prado Almeida et al. prepare lipid based nanoparticles which contained oleic acid was preferred as a liquid lipid due to its high solubility of the active substance (0,167 mg/mL) and it was reported that the formulations prepared with stearyl alcohol did not undergo phase separation (42).
It has been observed that LE solubility in surfactants is higher than in oils and lipids. LE showed high solubility in Lutrol® F68 (0.16328 mg/mL); subsequent to Labrasol® (0.12039 mg/mL), Tween® 80 (0.10116 mg/mL), Cremephor® RH40 (0.9983 mg/mL) and Cremephor® EL (0.9105 mg/mL). Following the solubility study in surfactants, conventional emulsions were prepared with surfactants. While no phase separation was observed in formulations using p as surfactant, phase separation was observed in other formulations. In a study by Leonardi et al., Lutrol F68 as surfactant and other surfactants (Tween® 80, Cremophor® A25, Lipoid® S100, and Kolliphor® HS) are present in varying proportions in the prepared SLN. It was observed that the lowest particle size and distribution of the prepared SLN formulations belonged to the formulation containing 2% (w/v) Lutrol® F68 (213.8, 0.182), while the formulation that maintained its physical stability for the longest time during storage was the formulations containing Lutrol® F68 (43). These findings show that Lutrol® F68 may be a suitable choice of surfactant for SLN and NLC formulations to be prepared. Generally, phase separation or aggregation was not encountered in all formulations prepared.
It was confirmed that optimal SLN and NLC formulations by choosing Lutrol® F68 as the surfactant, Compritol® 888 ATO, and cetyl stearyl alcohol as lipid and oleic acid as oil.
Measurement of LE amount and Analytical Method Validation
The retention time of LE was found to be 7.14 min (Fig. 1A). In the HPLC chromatogram obtained from the mobile phase, no peaks were observed in the mobile phase that could interfere with the LE peak (Figure 1B). In the peak of LE, there was no interference to the peaks of the other components used in the formulations and showing the specificity of the method (Figure 1). Linearity validation was performed with a standard calibration curve in the range 100–5000 ng/mL prepared from the stock solution of LE (1µg/mL). The equation of the regression created as a result of the calibration was calculated as y = 0.0253x + 0.2144 (r2=0.9906). It was observed that the relative standard deviations of the validation parameters of the method for accuracy, intra-day and inter-day precision were below 2%. While the recycling percentages of LE were determined as 93.57 ± 0.11 – 96.27 ± 0.19%, limit of detection (LOD) and limit of quantification (LOQ) were found to be 18.66 ng/mL and 54.82ng/mL, respectively.
Solubility Study
The solubility of LE in water and PBS was determined as 6.1913 ± 0.0008 µg/mL and 1.1122 ± 0.0005 µg/mL at 25 ± 1 oC, respectively.
Characterization of Formulations
Particle Size Analysis
Particle size analysis showed that formulations had particles in the range of 100–150 nm in average (Figs. 2 and 3). Polydispersity index (PI) values of less than 0.200 indicated that the formulations had a homogeneous and narrow particle size distribution (21, 22). The lowest particle size was obtained with formulation Comp-NLC (134.1 nm, 0.138 PI), while Comp-SLN (140.8 nm, 0.159 PI), Ca-NLC (141.7 nm, 0.199 PI) and Ca-SLN (143.9 nm, 0.168 PI) respectively followed this formulation. Incorporation of the drug did not remarkably increase the particle size of the formulations. It was obvious that the particle size of LE-Comp-NLC did not change significantly during 3 months of storage (p > 0.05), while the particle size of other formulations increased statistically (p ≤ 0.05). Formulation LE-CA-NLC displayed the highest increase from 143.9 nm (0.248 PI) to 155.6 nm (0.269 PI). Although statistical evaluations showed an increase in these formulations but not remarkable. No microparticle content or agglomeration was detected in all formulations during 3 months of storage.
In general, zeta potential of the formulations was between (-22.4) - (-24.8) and it did not significantly change over 3 months (p > 0.05). It was observed that the zeta potential of the drug loaded formulations was higher but exhibited insignificant difference compared to placebo formulations (p > 0.05).
SEM Analysis
SEM images provided information on the morphologies and particle sizes of CA-NLC, LE-CA-NLC, CA-SLN, LE-CA-SLN, Comp-NLC, LE-Comp-NLC, Comp-SLN and LE-Comp-SLN formulations. (Figure 4). When the DLS and SEM data were compared, it was concluded that they were consistent with each other, and it was proven that the lipid colloid systems did not contain micro-structured particles and crystallization signs of the active substance.
Encapsulation Efficiency and Drug Loading Capacity
EE of LE-CA-SLN, LE-CA-NLC, LE-Comp-SLN and LE-Comp-NLC was found to be 72.03 ± 0.78%, 77.82 ± 0.44%, 75.08 ± 0.22% and 79.03 ± 0.54%, respectively (Fig. 5). LC of formulations was 6.548 ± 0.0029%, 7.075 ± 0.0041%, 6.825 ± 0.008% and 7.185 ± 0.0017% in the same order. Type of surfactant, oil or solid lipid content, and amount of the drug have been stated as critical attributes that have impact on EE and LC of lipid nanoparticle formulations (22). Results demonstrated that oil amount was critical for EE and LC. The length of the behenic acid ester chain of Compritol® 888 ATO allowed more drug molecules to be trapped, resulting in higher EE (LE-Comp-NLC) compared to formulation containing cetyl stearyl alcohol (LE-CA-NLC) (37).
The negatively charged groups in the hydrocarbon tail of CA interact with the positive groups of the drug molecule and may have a negative effect on EE (37, 38).
The addition of liquid lipid to the formulation caused the ordered structure from solid lipid to become disordered. This irregular structure resulted in more drug entrapment in the formulation, resulting in an increase in EE and LC values. At the same time, the liquid lipid also reduces the crystallization that occurs in the formulation formed with the solid lipid, allowing the drug to be substituted in the formulation stably for a longer period of time. Developed as an alternative to SLN, NLC help the stable crystal structure of the solid lipid deteriorate and remain in the amorphous structure, helping the drug to be found in these amorphous structures (25).
Statistically, the results are demonstrated that, there is no significant difference on the EE and LC values of the formulations between PD and 3 months (p > 0.05). Unchanged EE and LC values indicated that the drug was not expulsed from the nanoparticles during storage.
FT-IR Analysis
FT-IR was used to see the physical and chemical interactions between the active ingredient and other ingredients. As shown in Figure 6, the intensity of characteristic spectra for LE was detected at much lower intensity and wider peaks than those caused by the other components. Tensile bands due to C=O vibrations arising from the ester structure in triglycerides caused sharp peaks to be seen at 1831.09–1559.54 cm− 1, 886.131-1289.18 cm− 1 and 743.224 cm− 1 (C–Cl stretch band). In addition to the bands originating from the vibrations of the acetyl group found in LE, bands originating from the C-H and O-H vibrations of the triglyceride group were detected at 2921.71–2818.96 cm− 1. Similar results have been reported in various studies (44–46). In formulations containing Compritol®888 ATO, two main peak spectra were observed at 1815 cm− 1 and 1706 cm− 1 due to the C=C stretching and the vibrations of the -OH group (C=C stretching and normal OH stretching, respectively) (47). In drug-loaded formulations, these peaks seem to disappear due to drug-related peaks (1831.09–1559.54 cm− 1).
For CA-SLN and CA-NLC the main peak for % transmittance is observed at 2800-2600 cm−1 which was identified to C–H group, however, this peak was observed as shifted at 2800-2600 cm−1 for LE-CA-SLN and LE-CA-NLC formulations. Another major peak for the bands from 1708–1738 cm−1, which corresponds to the C=O group, and from 1586–1604 cm− 1 corresponding to the C=C group, were observed in all the excipient spectra (48). Also, the peak between 870.06-610.18 cm− 1 (C‑O stretch) seen in all formulations except the pure LE is highly likely to be caused by Lutrol® F68. (49). It was concluded that there was no interaction between the active ingredient and excipients.
Thermal Behaviors of Formulations
The melting point of plain Compritol® 888 ATO was found as 74.94 oC with 225.16 J g− 1 melting enthalpy (Fig. 7) (Table III). Comp-SLN yielded a melting peak at 70.41 oC with a melting enthalpy of 37.526 J g− 1. CI was calculated as 50.81%. CA-SLN yielded a melting peak at 71.88 oC with a melting enthalpy of 29.833 J g− 1 due to the addition of surfactant to the dispersion (25). CI was calculated as 39.38%. The reduction in obtained initial and peak temperatures, which is evident in NLCs, may be due to the reduction in size. The fact that the melting range is lower than pure lipids can be explained by the transformation of the formulation towards an amorphous structure (50). The appearance of the amorphous structure can be attributed to the disruption of the ordered lattice structure of the solid lipid with much lower energy than the enthalpy that would be spent. Therefore, the result was obtained that the liquid lipid resulted in a lower lattice arrangement compared to the formulation containing the solid lipid. NLC formulations were attributed to the presence of liquid lipid incorporation resulting in delayed crystallization of nanoparticles. According to table 4, 67.58 oC and 27.772 J g− 1 71.82 oC melting enthalpies were obtained from Comp-NLC and LE-Comp-NLC. For formulations containing CA; 21.766 J g− 1 and 24.091 J g− 1 melting enthalpies were obtained from CA-NLC and LE-CA-NLC, resulting in 36.85 and 39.34% CI, respectively.
Incorporation of the drug in Comp-SLN induced the melting point and melting enthalpy. resulting in a remarkable decrease in CI. SLN based on CA, gave a double peak at 74.26 and 75.11 oC, which could be attributed to the mixture of cetyl alcohol and stearyl alcohol in CA (Fig. 7) (25). Similar behaviours were detected with CA based SLN formulations as can be seen in Table III.
Table III DSC parameters of formulations and formulation components after three months at room temperature.
Formulation
|
Storage
(RT)
|
Melting enthalpy ΔH (J/g)
|
Melting point (oC)
|
CI (%)
|
Bulk Cetyl stearyl alcohol
|
-
|
225.16
|
74.94
|
-
|
Bulk Compritol ATO888
|
-
|
192.23
|
78.23
|
-
|
CA-SLN
|
1st day
|
29.833
|
71.88
|
39.38
|
60th day
|
30.248
|
72.21
|
40.86
|
90th day
|
30.533
|
72.74
|
41.68
|
LE-CA-SLN
|
1st day
|
34.814
|
73.86
|
43.47
|
60th day
|
37.018
|
74.47
|
44.14
|
90th day
|
36.99
|
73.77
|
44.59
|
CA-NLC
|
1st day
|
21.766
|
70.47
|
36.85
|
60th day
|
22.912
|
71.13
|
37.72
|
90th day
|
25.942
|
71.44
|
37.91
|
LE-CA-NLC
|
1st day
|
24.091
|
72.91
|
39.34
|
60th day
|
26.259
|
72.96
|
39.93
|
90th day
|
26.295
|
71.29
|
40.16
|
Comp-SLN
|
1st day
|
37.526
|
70.41
|
50.81
|
60th day
|
37.564
|
71.38
|
51.07
|
90th day
|
37.640
|
72.09
|
51.56
|
LE-Comp-SLN
|
1st day
|
37.886
|
74.21
|
53.24a
|
60th day
|
38.050
|
72.38
|
54.36b
|
90th day
|
38.110
|
72.56
|
54.73c
|
Comp-NLC
|
1st day
|
23.427
|
67.58
|
43.39
|
60th day
|
22.65
|
69.62
|
44.8
|
90th day
|
26.685
|
68.5
|
45.13
|
LE-Comp-NLC
|
1st day
|
27.772
|
71.82
|
45.72
|
60th day
|
26.851
|
72.91
|
46.24
|
90th day
|
26.990
|
73.16
|
47.16
|
Cl: crystallinity indice |
In vitro Release Study
The cumulative percent release of LE from formulations (LE-Comp-SLN, LE-Comp-NLC, Control, LE-CA-SLN and LE-CA-NLC) was monitored for 8 hours. Each sample was analyzed six times. The outcomes are demonstrated in Fig. 8. It was found that, the concentration of the drug in the formulation directly affected the release rate. LE-Comp-SLN, LE-Comp-NLC, LE-CA-SLN and LE-CA-NLC displayed sustained drug release. LE release was the lowest with SLN followed by NLC as can be seen in Figure 7 (p < 0.05). The release rate of LE from the different formulations was 1.731 ± 0.017, 2.295 ± 0.015 2.389 ± 0.019 and 4.186 ± 0.022 mcg/mL/h. It was found out that Control LE solution released 100% of LE at the end of 2h while LE-Comp-NLC, LE-CA-NLC, LE-Comp-SLN and LE-CA-SLN released 69.71%. 64.11%. 64.06% and 59.51% of the drug respectively. In the control group, there was a significant difference between SLN and NLC formulations, as well as between SLN and NLC formulations. (p<0.05). In general, it was found that the release rate of LE increased due to the decrease in the %CI values of SLN and NLC (16,17). Addition of liquid lipid to SLN based on CA (LE-CA-NLC) brought out more shortcomings compare to SLN based on Compritol®888 ATO (LE-Comp-SLN).
The release profiles of the formulations were found to be in accordance with the Korsmeyer-Peppas release model (Table IV). In addition, since the n value (0.442) calculated in the Korsmeyer-Peppas model was lower than 0.45, it was found to be suitable for Fickian diffusion release (state I diffusional) for LE-CA-SLN (Table IV) (17.25). Since the n values of LE-Comp-NLC and LE-CA-NLC formulations (0.4792 and 0.4738, respectively) were between 0.45≤ n˂ 0.89, both diffusional and erosional drug release was observed and proved to be suitable for non-Fickian release. Considering the evaluations of LE-Comp-NLC and LE-CA-NLC formulations in terms of both in vitro release study and release kinetics, it is clear that the way of drug encapsulation results in amorphous or defective drug structure. Beside of that drug release from LE-Comp-SLN and control followed Higuchi type kinetic model with non-Fickian anomalous transport as indicated by Korsmeyer-Peppas model.
Table IV: Mathematical assessment of drug release from formulations.
Formulations
|
Zero order. R
|
First order. R
|
Hixson-Crowell. R
|
Higuchi. R
|
Korsmeyer-Peppas. R (n)
|
LE-Comp-SLN
|
0.9338
|
0.8253
|
0.9103
|
0.9957
|
0.9946 (0.4571)
|
LE-Comp-NLC
|
0.9486
|
0.8529
|
0.9227
|
0.9841
|
0.9982 (0.4792)
|
LE-CA-SLN
|
0.9266
|
0.8127
|
0.9055
|
0.9902
|
0.9932 (0.4421)
|
LE-CA-NLC
|
0.9471
|
0.8463
|
0.9108
|
0.9812
|
0.9973 (0.4738)
|
Control
|
0.7386
|
0.6944
|
0.9016
|
0.9605
|
0.9117 (0.3612)
|
Ex vivo Permeation Study
SLNs and NLCs were reported as penetration enhancers in several studies (17). SLN and NLC formulations were increased the penetration rate of LE when compared with the control group (p≤0.05). There is no remarkable difference between LE-Comp-SLN and LE-CA-SLN formulations (p>0.05). The steady-state flux achieved by drug penetration was different between the control LE and nanoparticles. It was observed between 2- 6 h for LE-Comp-SLN, LE-Comp-NLC, LE-CA-SLN and LE-CA-NLC and between 0- 4 hours for control LE. The steady-state flux achieved by drug penetration was different between the control LE and nanoparticles. It was observed between 4 and 8 hours for LE-Comp-SLN, LE-Comp-NLC, LE-CA-SLN and LE-CA-NLC and between 1 and 4 hours for control LE. In those periods steady state flux values were 52.998 µg.cm−2 h−1. 57,835 µg.cm−2 h−1. 52,109 µg.cm−2 h−1. 53,232 µg.cm−2 h−1. and 26,623 µg.cm−2 h−1 LE-Comp-SLN, LE-Comp-NLC, LE-CA-SLN, LE-CA-NLC and control, respectively (Fig. 9). It is observed that there is a statistically significant difference the flux value of the LE-Comp-NLC formulation compared to LE-Comp-SLN, LE-CA-SLN, LE-CA-NLC and control group (p ≤ 0.05).
Tape Stripping Study
Extraction of the drug from the porcine skin followed by tape stripping and skin homogenization process was performed for nanoparticles and control group; the results are showed in Fig. 10. The values of LE obtained from LE-Comp-SLN, LE-Comp-NLC, LE-CA-SLN, LE-CA-NLC and control were 0.1699 mg (2.53%), 0.1617 mg (2.11%), 0.1661 mg (2.88%), 0.1632 (2.38%) and 6.693 mg (67.79%) in the SC and 3.236 mg (47.16%), 3.985 mg (49.11%), 3.112 mg (45.29%), 2.666 mg (46.62%) and 0.243 mg (2.43%) in the viable skin layers (epidermis and dermis) and 3.928 mg (52.36%), 4.083 mg (51.67%), 3.690 mg (47.42%), 3.699 mg (47.42%) and 2.971 mg (29.22%) in the donor compartment. respectively. It was observed that there was no statistically significant difference between LE-Comp-SLN, LE-Comp-NLC, LE-CA-SLN and LE-CA-NLC in terms of LE amount in SC (p > 0.05). Besides of that, when examined in terms of the amount of cumulative LE in donor phase and the amount of LE in viable dermis, it is seen that LE-Comp-NLC has statistically significant difference from other formulations (p ≤ 0.05).
Overall NLC formulations exhibited higher penetration enhancer effect than SLN. LE-Comp-NLC exhibited the highest retention of LE in the deeper parts of the skin. For LE-Comp-NLC formulation 49.11% of the total amount of LE that penetrated from the skin was held in the epidermis and dermis. This amount was remarkably higher than control group (p ≤ 0.05) but not significant with LE-CA-NLC and LE-CA-SLN formulations (p>0.05).
Additionally, Compritol® 888 ATO based nanoparticles escaped to the deeper layers due to advantageous nanometer size. Moreover, spontaneous occlusion of lipid nanoparticles contributed to the penetration properties through the skin. Subsequent skin hydration can be a reason of promoted drug penetration (25, 51).