Table 4.

Physicochemical and biophysical characterization of empty DPPC liposomes and DPPC liposomes loaded with TDF by different methods (incubation, hydration, and direct mixing), including: Encapsulation efficiency (EE%), mean hydrodynamic diameter (Z-average), polydispersity index (PDI), zeta potential, main phase transition temperature (T_m), and cooperativity of the phase transition (B). R² is the coefficient of determination of T_m and B after fitting data with Equation (1).

Type of Characterization	Parameters Evaluated	DPPC	DPPC+TDF
			Incubation	Hydration	Direct Mixing
Physicochemical	EE (%)	-	84.63 ± 5.09 ns	87.31 ± 4.68 ns	84.79 ± 6.51 ns
	Z-average (nm)	123.97 ± 3.62	113.13 ± 0.38 ns	113.47 ± 1.07 **	129.83 ± 6.69 **
	PDI	0.11 ± 0.01	0.06 ± 0.01 **	0.10 ± 0.01 ***	0.24 ± 0.01 ***
	Zeta potential (mV)	−3.55 ± 0.29	+6.00 ± 0.28 ***	+0.80 ± 0.33 ***	−5.29 ± 0.280 ns
Biophysical	Tm	41.05 ± 0.03	41.19 ± 0.05 ns	41.27 ± 0.02 ns	41.49 ± 0.03 ns
	B	2781 ± 234	4117 ± 724 ns	3922 ± 238 ns	3135 ± 298 ns
	R2	0.999	0.999	0.999	0.999

Results are the mean ± standard deviation of at least three independent measurements, and comparisons were performed using one-way ANOVA with the Tukey–Kramer post-test for the following paired observations: 1) EE% obtained with the incubation method vs. EE% obtained with the hydration method; EE% obtained with the hydration method vs. EE% obtained with the direct mixing method; EE% obtained with the direct mixing method vs. EE% obtained with the incubation method; 2) Z-average, PDI, zeta potential, T_m, or B of: liposomes of DPPC loaded with TDF by the incubation method vs. liposomes of DPPC loaded with TDF by the hydration method; liposomes of DPPC loaded with TDF by the hydration method vs. liposomes of DPPC loaded with TDF by the direct mixing method; liposomes of DPPC loaded with TDF by the direct mixing method vs. liposomes of DPPC loaded with TDF by the incubation method; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant.