2.2.1 Micelle and hydrogel preparation
Poloxamers (PLs) were acquired from Sigma-Aldrich. For micellar solution preparation, 3% (m/v) PL 407, alone or in a binary system with PL 403, was solubilized in ultrapure water and kept at 4 ºC while stirred magnetically (150 rpm) until a transparent solution was obtained (Schmolka, 1972). In the same manner, for hydrogel preparation, two formulations were standardized: formulation 1 (F1), composed of 30% PL407 and formulation 2 (F2), 2% PL403 and 28% PL407 PL were dispersed in deionized water until they were complete dissolved.(Akkari et al ., 2016) The antibodies were dispersed into the formulations at a final concentration of 0.1% (m/v). The formulations were stored at 4 ºC until use.
2.2.2 Micellar hydrodynamic diameter: antibody-micelle interaction
The micellar hydrodynamic diameter and the size average distribution were determined using a Zetasizer ZS particle analyzer (Malvern Instruments Ltd.) at a fixed angle of 173º and temperatures of 25 ºC and 32.5 ºC to simulate the micelle behavior at room temperature and physiological skin temperature. All samples were analyzed before and after antibody incorporation. All measurements were repeated at least three times for each sample.
Rheological analysis
Rheological analysis was carried out on a Kinexus rotational rheometer (Malvern Instruments Ltd.), using cone-plate geometry (40 mm diameter size). For the sol-gel transition temperature (Tsol-gel) determination, measurements were performed at a temperature that ranged from 10 to 50 ºC using a sample volume of 1 mL, a gap between the plates of 1 mm, a frequency of 1 Hz and shear stress of 2 Pa. Additionally, a frequency sweep analysis ranging from 0.1 to 10 Hz (at 32.5 °C) was performed. All measurements were used to determine the parameters related to the elastic modulus (G´), the viscous modulus (G“) and viscosity (η). RSpace for Kinexus software was used for the data analysis.