Figure 1.
Experimental design for the in vitro analyses. Day 1: B16-F10 cells were cultivated for 24 h. Day 2: B16-F10 cells were washed in PBS and then treated with PDT. In the SLN-AlPc treatment, B16-F10 were treated and then irradiated for 10 min (660 m, 25.88 J/cm2). Day 3: Different analyses were carried out 24 h after the treatment.
Figure 1.
Experimental design for the in vitro analyses. Day 1: B16-F10 cells were cultivated for 24 h. Day 2: B16-F10 cells were washed in PBS and then treated with PDT. In the SLN-AlPc treatment, B16-F10 were treated and then irradiated for 10 min (660 m, 25.88 J/cm2). Day 3: Different analyses were carried out 24 h after the treatment.
Figure 2.
Images obtained with the technique of polarized light microscopy with 40× magnification of the butters: (A) murumuru; (B) babassu; (C) bacuri; (D) ucuuba.
Figure 2.
Images obtained with the technique of polarized light microscopy with 40× magnification of the butters: (A) murumuru; (B) babassu; (C) bacuri; (D) ucuuba.
Figure 3.
Response surface graphs obtained after BB analysis of the variables: HD (A), PdI (B), and ZP (C).
Figure 3.
Response surface graphs obtained after BB analysis of the variables: HD (A), PdI (B), and ZP (C).
Figure 4.
In vitro skin permeation profile of AlPc and SLN-AlPc over 24 h.
Figure 4.
In vitro skin permeation profile of AlPc and SLN-AlPc over 24 h.
Figure 5.
Transmission electron microscopy images of SLN-AlPc (A,B) and SLN (C). The micrographs show a spherical shape with small dark spots (A,B), suggesting the presence of AlPc. AlPc has a high affinity for osmium tetroxide, which explains the darker labeling. It is possible to observe that the SLN (C) presents a different morphology.
Figure 5.
Transmission electron microscopy images of SLN-AlPc (A,B) and SLN (C). The micrographs show a spherical shape with small dark spots (A,B), suggesting the presence of AlPc. AlPc has a high affinity for osmium tetroxide, which explains the darker labeling. It is possible to observe that the SLN (C) presents a different morphology.
Figure 6.
SERS spectra of (a) SLN-AlPc formulation, (b) physical mixture (AlPc + SLN), (c) solution of AlPc in alcohol, and (d) SLN solution of AlPc in alcohol.
Figure 6.
SERS spectra of (a) SLN-AlPc formulation, (b) physical mixture (AlPc + SLN), (c) solution of AlPc in alcohol, and (d) SLN solution of AlPc in alcohol.
Figure 7.
Internalization of SLN-AlPc. The fluorescence histogram showed the internalized amount of SLN-AlPc at different time points after the SLN-AlPc treatment. The fluorescence signal increases proportionally to the time that the B16-F10 were exposed to SLN-AlPc.
Figure 7.
Internalization of SLN-AlPc. The fluorescence histogram showed the internalized amount of SLN-AlPc at different time points after the SLN-AlPc treatment. The fluorescence signal increases proportionally to the time that the B16-F10 were exposed to SLN-AlPc.
Figure 8.
(A) Ultrastructure of B16-F10 treated with SLN-AlPc for 15 min. (B) Internalized aggregated SLN-AlPc is present in B16-F10 (*) and in a liposome (L). Melanosoma is present in the melanocyte cells (slashed box). Nucleus (N), mitochondria (M), and cytoplasm (C) are indicated in the image.
Figure 8.
(A) Ultrastructure of B16-F10 treated with SLN-AlPc for 15 min. (B) Internalized aggregated SLN-AlPc is present in B16-F10 (*) and in a liposome (L). Melanosoma is present in the melanocyte cells (slashed box). Nucleus (N), mitochondria (M), and cytoplasm (C) are indicated in the image.
Figure 9.
Internalization of SLN-AlPc after 5, 15, and 30 min of treatment. The nucleus (blue), the SLN-AlPc (green), and the AlPc (red) are demonstrated in the image.
Figure 9.
Internalization of SLN-AlPc after 5, 15, and 30 min of treatment. The nucleus (blue), the SLN-AlPc (green), and the AlPc (red) are demonstrated in the image.
Figure 10.
Endocytosis pathway characterization using different endocytosis inhibitors: (A) a reduction in the fluorescence signal of SLN-AlPc was observed when treated with amiloride, indicating a decrease in SLN-AlPc internalization by the macropinocytosis pathway; (B) histogram of the fluorescence signal in B16-F10 cells treated with amiloride and SLN-AlPc. The cytochalasin D (C), nystatin (D), phenylarsin (E), and sodium azide (F) did not alter the fluorescence signal of SLN-AlPc.
Figure 10.
Endocytosis pathway characterization using different endocytosis inhibitors: (A) a reduction in the fluorescence signal of SLN-AlPc was observed when treated with amiloride, indicating a decrease in SLN-AlPc internalization by the macropinocytosis pathway; (B) histogram of the fluorescence signal in B16-F10 cells treated with amiloride and SLN-AlPc. The cytochalasin D (C), nystatin (D), phenylarsin (E), and sodium azide (F) did not alter the fluorescence signal of SLN-AlPc.
Figure 11.
Colocalization assays of SLN-AlPc. The figure shows the colocalization of fluorescence relative to AlPc for cytoskeleton (A,D,G,J), nucleus (B,E,H,K), and fluorescence relative to SLN-AlPc (C,F,I,L). The white dots in the figures represent positive fluorescence pixels, indicating colocalization. In the last column, the colocalization index is shown in %, in addition to the Pearson coefficient that was used for the analyses.
Figure 11.
Colocalization assays of SLN-AlPc. The figure shows the colocalization of fluorescence relative to AlPc for cytoskeleton (A,D,G,J), nucleus (B,E,H,K), and fluorescence relative to SLN-AlPc (C,F,I,L). The white dots in the figures represent positive fluorescence pixels, indicating colocalization. In the last column, the colocalization index is shown in %, in addition to the Pearson coefficient that was used for the analyses.
Figure 12.
The cytoskeleton of B16-F10 cells. Macropynosomes (*) can be observed in B16-F10 cells treated with SLN-AlPc. In blue, the nucleus (N) can be observed.
Figure 12.
The cytoskeleton of B16-F10 cells. Macropynosomes (*) can be observed in B16-F10 cells treated with SLN-AlPc. In blue, the nucleus (N) can be observed.
Figure 13.
Cellular viability of B16-F10 cells treated with SLN-AlPc. The percentage of B16-F10 cell viability was calculated at different concentrations of SLN-AlPc treatment (A,B). The percentage of B16-F10 cell viability was calculated at different treatment conditions using propidium iodide (C). The absorbance of violet crystal was calculated under different treatment conditions to evaluate the cell death (D) *** p < 0.001 and **** p < 0.0001 compared to control.
Figure 13.
Cellular viability of B16-F10 cells treated with SLN-AlPc. The percentage of B16-F10 cell viability was calculated at different concentrations of SLN-AlPc treatment (A,B). The percentage of B16-F10 cell viability was calculated at different treatment conditions using propidium iodide (C). The absorbance of violet crystal was calculated under different treatment conditions to evaluate the cell death (D) *** p < 0.001 and **** p < 0.0001 compared to control.
Figure 14.
ROS production in B16-F10 cells treated with SLN-AlPc. Images were taken in confocal microscopy (A,B) and paramagnetic resonance (C). (A) B16-F10 cells that were not irradiated had no ROS production (green) since only the nucleus (blue) could be seen in the image. (B) ROS production was observed after B16-F10 irradiation. (C) Quantification of ROS production in different treatment conditions. **** p < 0.0001 compared to control.
Figure 14.
ROS production in B16-F10 cells treated with SLN-AlPc. Images were taken in confocal microscopy (A,B) and paramagnetic resonance (C). (A) B16-F10 cells that were not irradiated had no ROS production (green) since only the nucleus (blue) could be seen in the image. (B) ROS production was observed after B16-F10 irradiation. (C) Quantification of ROS production in different treatment conditions. **** p < 0.0001 compared to control.
Figure 15.
B16-F10 cells morphology in TEM and SEM after the PDT treatment with SLN-AlPc; it was also possible to observe formation of apoptotic bodies (*) and cytoplasmic bumps.
Figure 15.
B16-F10 cells morphology in TEM and SEM after the PDT treatment with SLN-AlPc; it was also possible to observe formation of apoptotic bodies (*) and cytoplasmic bumps.
Figure 16.
Analysis of apoptotic cell death. Apoptosis was evaluated by Western blot (A–F) and flow cytometry (G–J). Western blot images of Bax (A), Bcl2 (C), and caspase 3 (E) bands as well as the loading control (beta acting) bands. Pixel quantification of Bax (B), Bcl2 (D), and caspase 3 (F) bands to the loading control bands. Apoptosis was induced by phosphatidylserine under different treatment conditions (G–J). Q1 represents the Propidium Iodine positive signal, which is a marker for necrosis. Q2 represents both propidium iodide (necrose) and Annexin-V (Apoptose) positive signal. Q3 represents only Annexin-V positive signal, and Q4 represents no positive signal and, therefore, no cell death.
Figure 16.
Analysis of apoptotic cell death. Apoptosis was evaluated by Western blot (A–F) and flow cytometry (G–J). Western blot images of Bax (A), Bcl2 (C), and caspase 3 (E) bands as well as the loading control (beta acting) bands. Pixel quantification of Bax (B), Bcl2 (D), and caspase 3 (F) bands to the loading control bands. Apoptosis was induced by phosphatidylserine under different treatment conditions (G–J). Q1 represents the Propidium Iodine positive signal, which is a marker for necrosis. Q2 represents both propidium iodide (necrose) and Annexin-V (Apoptose) positive signal. Q3 represents only Annexin-V positive signal, and Q4 represents no positive signal and, therefore, no cell death.
Table 1.
Independent variables selected for Box–Behnken method.
Table 1.
Independent variables selected for Box–Behnken method.
Independent Variables | | Levels | |
---|
−1 | 0 | +1 |
---|
A: Butter: Surfactant | 1:1 | 1:1.5 | 1:2 |
B: AlPc (μM) | 20 | 40 | 80 |
C: Temperature (°C) | 75 | 80 | 85 |
Table 2.
Combinations generated from independent variables.
Table 2.
Combinations generated from independent variables.
Number of Experiment | A | B | C |
---|
1 | 0 | 1 | −1 |
2 | −1 | 0 | 1 |
3 | −1 | −1 | 0 |
4 | 0 | −1 | −1 |
5 | 0 | 0 | 0 |
6 | 0 | 0 | 0 |
7 | 1 | 0 | 1 |
8 | −1 | 0 | −1 |
9 | 0 | −1 | 1 |
10 | −1 | 1 | 0 |
11 | 1 | −1 | 0 |
12 | 1 | 1 | 0 |
13 | 0 | 0 | 0 |
14 | 1 | 0 | −1 |
15 | 0 | 1 | 1 |
Table 3.
Endocytosis inhibitors and their respective target pathways.
Table 3.
Endocytosis inhibitors and their respective target pathways.
Inhibitor | Concentration | Inhibition Pathway |
---|
Sodium azid | 100 mM | ATP-dependent pathways |
Amiloride | 0.2 mM | Macropinocytosis |
Cytochalasin D | 1 μM | Pinocytosis |
Phenylarsine | 0.2 μM | Clathrin-mediated pathway |
Nystatin | 20 μg/mL | Caveolin-mediated pathway |
Table 4.
HD, PdI, and ZP obtained with previous SLNs.
Table 4.
HD, PdI, and ZP obtained with previous SLNs.
Sample | HD (nm) | PdI | ZP (mV) |
---|
SLN Murumuru | 27.5 ± 0.2 | 0.191 ± 0.001 | −11.80 ± 2.60 |
SLN Babaçu | 100.6 ± 0.2 | 0.266 ± 0.005 | −10.40 ± 0.15 |
SLN Bacuri | 536.4 ± 49.8 | 0.600 ± 0.039 | −22.30 ± 1.18 |
SLN Ucuuba | 140.8 ± 2.7 | 0.526 ± 0.006 | −21.60 ± 0.12 |
Table 5.
HD, PdI, ZP, and EE% values before and after BB optimization.
Table 5.
HD, PdI, ZP, and EE% values before and after BB optimization.
Sample | HD (nm) | PdI | ZP (mV) | EE% |
---|
SLN | 55.53 ± 0.23 | 0.191 ± 0.001 | −11.80 ± 2.60 | 61.50 ± 0.67 |
SLN optimized | 17.64 ± 0.21 | 0.173 ± 0.013 | −5.61 ± 0.93 | 66.40 ± 1.12 |
Table 6.
Results were obtained with DSC analysis of murumuru butter, AlPc, and SLN.
Table 6.
Results were obtained with DSC analysis of murumuru butter, AlPc, and SLN.
Sample | Melting Point (°C) | ΔH (Jg−1) |
---|
Murumuru butter | 38.4 | 63.9 |
AlPc | 175.1 | 194.3 |
SLN | 96.3 | 1036.0 |
SLN-AlPc | 93.3 | 873.2 |