pH-sensitive niosomes: Effects on cytotoxicity and on inflammation and pain in murine models

Figures & data

Table 1. Sample composition (in bold: selected formulations).

Sample	TW20(mM)	TW20-GLY(mM)	CHOL(mM)	IBU(%p/v)	LIDO(%p/v)
TW20	15	–	15	–	–
TW20 1% IBU	15	–	15	1	–
TW20 3% IBU	15	–	15	3	–
TW20 5% IBU	15	–	15	5	–
TW20 7% IBU	15	–	15	7	–
TW20 1% LID	15	–	15	–	1
TW20 3% LID	15	–	15	–	3
TW20 5% LID	15	–	15	–	5
TW20 7% LID	15	–	15	–	7
TW20-GLY	3.75	11.25	7.5	–	–
TW20-GLY 1% IBU	3.75	11.25	7.5	1	–
TW20-GLY 3% IBU	3.75	11.25	7.5	3	–
TW20-GLY 5% IBU	3.75	11.25	7.5	5	–
TW20-GLY 7% IBU	3.75	11.25	7.5	7	–
TW2OGLY 1% LID	3.75	11.25	7.5	–	1
TW20-GLY 3% LID	3.75	11.25	7.5	–	3
TW20-GLY 5% LID	3.75	11.25	7.5	–	5
TW20-GLY 7% LID	3.75	11.25	7.5	–	7

Table 2. Sample characterization.

Sample	Size (nm)	Potential, ζ (mV)	PDI	Fluorescence anisotropy	EE (mg/ml)
TW20	140.8 ± 1.6	−30.4 ± 1.0	0.219 ± 0.04	0.21 ± 0.01	–
TW20 5% IBU	90.6 ± 1.8	−31.7 ± 0.3	0.306 ± 0.09	0.30 ± 0.02	0.28 ± 0.04
TW20 5% LIDO	99.3 ± 1.8	−28.9 ± 0.1	0.133 ± 0.09	0.29 ± 0.01	7.00 ± 0.03
TW20-GLY	215.0 ± 3.0	−41.0 ± 1.2	0.160 ± 0.08	0.17 ± 0.01	–
TW20-GLY IBU 5%	122.1 ± 19.6	−40.2 ± 0.1	0.404 ± 0.05	0.20 ± 0.04	0.18 ± 0.05
TW20-GLY LIDO 5%	121.0 ± 2.3	−42.8 ± 1.3	0.203 ± 0.02	0.23 ± 0.02	8.65 ± 0.04

Figure 1. SAXS intensity spectra for Tw20 (left panel) and TW20-GLY (right panel) based systems, at pH 7.4, vertically shifted for enhanced visibility. From bottom to top: unloaded (black), loaded with 5% ibuprofen, loaded with 5% lidocaine. Dash lines decrease with q⁻² behavior, typical for bilayer structures.

Figure 2. Release profiles of IBU and LID from the vesicular carriers in HEPES (pH =7.4) at 32 °C as a function of time: (A) TW20 samples; (B) TW2–0GLY samples. Release experiments were carried out in triplicate. The reported value represents mean values and lay within 10% of the mean.

Figure 3. Cytotoxic effects of TW20 and TW20-GLY on Balb-3T3 cells as evaluated by CFE assay. Cells were exposed to increasing concentrations (0.1–100 μM) of Tw20 formulation and to the same concentration of Tween 20 control for 2 h (A) and 24 h (B) and to increasing concentrations (0.1–100 μM) of TW20GLY formulation and of Tween-20 glycine control not in vesicular form for 2 h (C) and for 24 h (d). In this range of concentrations, statistically significant cytotoxicity was found in Balb/3T3 cells exposed for 24 h to Tween-20 control at 50 μM and 100 μM (***p < 0.0001) but not to the Tw20 vesicles at the same concentrations. Statistically significant cytotoxicity was also found in cells exposed to Tw20GLY formulation at 50 μM (**p < 0.001) and 100 μM (***p < 0.0001) but not to the Tween-20 glycine control at the same concentrations.

Figure 4. Cytotoxic effects of TW20 and TW-20GLY on HaCaT cells evaluated by CFE assay. HaCaT cells were exposed to increasing concentration of TW20 formulation and to the same concentration of Tween-20 control for 2 h (A) and 4 h (B) and to increasing concentrations (0.1–100 μM) of TW20-GLY formulation and of Tween-20 glycine control for 2 h (C) and for 24 h (d). In this range of concentrations, statistically significant cytotoxicity was observed only in HaCat cells exposed to TW20 formulation at 50 and 100 μM (**p < 0.001).

Figure 5. In vivo effects of drug-loaded nonionic surfactant vesicles on formalin-induced nociception (IBU: panel A; LID: panel B). Purified formulation of vesicles and drug solution at the same drug concentration were used. The data are considered to be statistically significant for *Pb0.05, **Pb0.01 and ***Pb0.001 versus vehicle-treated animals (HEPES buffer). N = 9–10.

Figure 6. In vivo effects of drug-loaded vesicles in edema induced by zymosan. Purified formulation of vesicle and drug solution at the same drug concentration were used. The data are considered to be statistically significant for *Pb0.05, **Pb0.01 and ***Pb0.001 versus vehicle-treated animals (HEPES buffer). N = 10–12.