*2.5. DEX Quantification*

DEX was quantified through HPLC, in terms of encapsulation efficiency (*%EE*), according to an indirect method (Equation (1)):

$$\%EE = \frac{\mathcal{W}\_T - \mathcal{W}\_{\text{NE}}}{\mathcal{W}\_T} \times 100,\tag{1}$$

where *WNE* is the amount of DEX quantified in the filtrate (DEX not encapsulated) and W<sup>T</sup> is the DEX quantified in the total formula. The LPNCs were centrifuged in 100 KDa amicon ultra (Merck Millipore, Barcelona, Spain) at 4500 rpm for 30 min. DEX in the filtrate and in LPNCs (total drug content) were analyzed using a High-Performance Liquid Chromatograph (HPLC) Alliance, with a photodiode array detector (PDA). A C18 column (250 × 4.6 mm, 3 µm) was used with an isocratic mixture of acetonitrile:KH2PO<sup>4</sup> 0.05 M buffer (60:40) as the mobile phase, with a flow rate of 1.8 mL/min and an injection volume of 20 µL. The sample and column temperatures were 25 ◦C.

The amount of total drug entrapped per weight of nanoparticle or loading capacity (*%LC*) was calculated using the following equation:

$$\% \text{LC} = \% \text{EE} \times \frac{M\_{\text{DEX}}}{M\_{\text{LPNCs}}} \text{'} \tag{2}$$

where *MDEX* is the amount of active ingredient initially added to the formulation and *MLPNC*<sup>s</sup> is the mass of the nanoparticle components.
