Nucleopore track etch

For focal electroporation on slices, acute coronal slices of E14.5 Htr3a-GFP⁺ embryos were transferred on membranes (nucleopore Track-Etch; 1 μm pore size, Whatman) floating on NBM in an incubator (37 °C, 5% CO₂) for at least 2 h following Vibratome sectioning. A plasmid encoding for a red fluorescent protein (pUBI-tdTOM) was focally injected at the concentration of 1 μg ml⁻¹ through a beveled glass micropipette into the MGE using a nanoinjector (B203XVZ; WPI) and precision of the injection site was controlled by addition of 10% Fast Green (Sigma) to the plasmid. The slices were kept moisturized in HBSS and electroporated between platinum Petri dish and cover square platinum electrodes (CUY701-P5E and CUY701-P5L; Nepa Gene) with two unipolar square pulses (ΔP: 100 V; duration: 5 ms; interval: 500 ms) generated by a CUY21-SQ electroporator (Nepa Gene). Slices were then cultured 3 days in vitro (+DIV3) in an incubator (37 °C and 5% CO₂).

The in vitro release of TTC, free (in solution) or encapsulated by NLC (CP-TRANS/TTC, CP-DK/TTC, MM-DK/TTC), was measured using a Franz vertical diffusion cell system under sink condition^{27 (link)}. The test samples (0.4 mL) were placed in the donor compartment of the diffusion cells, which was separated by a polycarbonate membrane (Nucleopore Track-Etch, 0.1 mm pore size, Whatman)^{28 (link)} from the acceptor compartment containing 4 mL of the release medium (5 mM PBS, pH 7.4 with 5% Tween 80). The system was kept at 37 °C under magnetic stirring (300 rpm). At predetermined intervals during 50 h, aliquots (0.2 mL) were extracted from the acceptor compartment and the volume was replaced with the release medium. The concentration of released TTC in the aliquots was determined by HPLC (n = 5).
The release curves were analyzed with the KinetDS 3.0 software^{29 (link)}. Several kinetic models were tested and according to the coefficient of determination (R²) the best fit for NLC formulation curves was reached with the Korsmeyer–Peppas model: $$Q=k·t^n$$ where Q is fraction of drug released at time t, k is the rate constant, and n is the release exponent that typifies the drug release mechanism: n = 0.43 indicates Fickian diffusion, n = 1 means zero-order release while 0.43 < n < 1 values are related to anomalous transport^{30 (link)}.