2.5. Characterization

FTIR was performed on an IRAffinity Shimadzu spectrometer (Shimadzu Scientific Instruments, Columbia, MD, USA) in a 4000–400 cm⁻¹ wavelength range, with an accumulation of 32 scans and resolution of 4 cm⁻¹. In order to prepare the pellets, the sample and KBr were dried at 60 °C for 8 h and mixed in a ratio of 1:100 (sample:KBr).

A Bruker AVANCE III spectrophotometer 400 MHz (Bruker, Billerica, MA, EUA) was used to obtain ¹H NMR spectra, aiming the samples’ characterization. Basically, the Equations (1)–(3) are used to determine the average degree of acetylation of chitosan (DA¯) ([50,51]), degree of substitution (DS¯) of the derivative 3,6-O,O’-myristoyl chitosan ([37]) and degree of quaternization (DQ¯) of the derivative N-(2-hydroxy)-propyl-3-trimethylammonium, O-mysristoyl chitosan ([46]), respectively. In other words, DA¯, DS¯, and DQ¯ refers to the amount of acetylated units present in chitosan, as well as both the myristoyl groups added to chitosan and the quaternary groups present in derivative N-(2-hydroxy)-propyl-3-trimethylammonium,O-mysristoyl chitosan.

A_CH3 = area of the methyl hydrogens of the acetamido group (of GlcNAc units);

A_H2–H6 = area of hydrogens bonded to the C (2–6) carbon of the glycopyranose ring.

A_Me = area of the methyl hydrogens of the myristoyl group;

A_H2–H6 = area of hydrogens bonded to the C (2–6) carbon of the glycopyranose ring.

I_H1′= the integral of the signal due to the anomeric hydrogen bonded to N-substituted GlcN (units);

I_H1 = is the integral of the signal due to the anomeric units hydrogen bonded to unsubstituted GlcN (units).

The DQ¯ of DMcat samples were also determined by dosing the counter-ions Cl⁻ through titration with standardized 0.017 mol L⁻¹ aqueous AgNO₃ solution. Thus, the quaternized derivative (0.1 g) was dissolved in deionized water (100 mL) and the conductivity of the solution was measured at 25.00 ± 0.01 °C as a function of the added volume of aqueous AgNO₃ by using a Handylab LF1 conductivimeter (SI Analytics, Mainz, Germany). The value of DQ¯ of samples was calculated from the titration curves according to [46].

The conductivity method was performed to determine the CAC by using a Consort C863 conductivity meter. In order to prepare the samples, different polymer concentrations were utilized (1 × 10⁻⁶ mg/mL ≤ Cp ≤ 1 mg/mL). The DMCh sample was solubilized in acid medium due to its limited solubility in neutral medium, while deionized water was used to dissolve DMCat.

The average size, polydispersity, and zeta potential of DMCh (in acid medium) and DMCat (in water) micelles were determined using dynamic light scattering (DLS) measurements (ZetaSizer Nano ZS, Malvern, UK). Three replicates of each formulation were produced and analyzed.

The drug encapsulation efficiency (EE%) was quantified by measuring the absorbance of the CUR solution at 340 nm using a UV-Vis spectrophotometer [3,7]. The measurements were performed by an indirect method in triplicate, in which the unencapsulated drug was solubilized in acetone and quantified. The amount of CUR loaded into the DMCh and DMCat micelles was determined according to Equation (4):