Lipid Nanosystems and Serum Protein as Biomimetic Interfaces: Predicting the Biodistribution of a Caffeic Acid-Based Antioxidant

Figures & data

Figure 1 Chemical structures of caffeic acid (A) and mitochondriotropic antioxidant AntiOxCIN₃ (B).

Figure 2 (A) Fluorescence spectra of HSA (2.0×10⁻⁶ M) in the presence of increasing AntiOxCIN₃ concentrations (0 to 3.5×10⁻⁴ M) at 37 °C. (B) Deconvolution of HSA Trp (long dashes) and Tyr (short dashes) residues emission bands in the absence (pink) and in the presence (blue) of 3.5×10⁻⁴ M of AntiOxCIN₃. (C) Synchronous fluorescence spectra of HSA (2.0×10⁻² M) in absence and in presence of increasing concentrations of Anti-OxCIN₃ (0 to 3.50×10⁻⁴ M) with Δλ = 15 nm corresponding to Tyr residues and (D) Δλ = 60 nm corresponding to Trp residues.

Figure 3 Graphic representation of R_h obtained from DLS (A) and Zeta Potential values (B) for HSA and HSA-AntiOxCIN₃ ([AntiOxCIN₃] from 0 to 3.50×10⁻⁴ M), at three different temperatures represented by different colors and signs. The black dashed line shows the tendency for neutralization upon AntiOxCIN₃ addition observed by increase values of R_h and zeta potential.

Figure 4 PC membrane model system partition coefficient of AntiOxCIN₃ by derivative spectrophotometry: (A) Third derivative of AntiOxCIN₃ (2.0×10⁻⁴ M) absorption spectra in aqueous buffered phase pH=7.4 (red), after incubation with PC membrane model system (0–3.0×10⁻³ M) (black) and PC membrane model system in the absence of AntiOxCIN₃ (grey); (B) Non-linear fitting by Equation 1(1) of derivative absorbance at λ=341 nm as a function of PC membrane model system concentration. (C) Third derivative of the absorbance spectra and respective non-linear fitting by Equation 1(1) (dashed line) at several temperatures; (D) van’t Hoff plot representing the Gibbs free energy values as function of the inverse of temperature, in K⁻¹, with the transition temperature between gel (L_β’) and fluid (L_α) lipid phases highlighted.

Figure 5 (A) Stern-Volmer plots of the probes n-AS incorporated in the PC membrane system at 37 °C as a function of AntiOxCIN₃ ([AntiOxCIN₃]_M). Different colors and signs represent the different probes used. The full line represents the linear fitting applied to steady-state data. The dashed line represents the linear fitting applied to the data obtained from lifetime measurements. (B) Schematic representation of the possible location of the n-AS (n=3, 6, 9 and 12) fluorescent probes and AntiOxCIN₃ within PC molecules of the PC membrane system. Membrane microviscosity assessment studies in the presence (red) and absence (black) of AntiOxCIN₃ and respective sigmoidal fits: (C) Anisotropy, r_ss, profile of DPH probe in the PC membrane model system dependent on the temperature with fitting using Equation 3(3) ; (D) Sigmoidal profile of normalized mean count rate (MCR) obtained by DLS as function of temperature with sigmoidal fit using Equation 14(14) .

Table 1 Fluorescence Quenching Constants Obtained from Measurements of Fluorescence Quenching Studies for n-AS Fluorescence Probes in PC Membrane Model System at 37°C

Probe	3-AS	6-AS	9-AS	12-AS
KSV (M^−1)	11.25	1.54	2.99	2.90
Kq (M^−1∙s^−1)	1.36 × 10^6	1.73 × 10^5	2.71 × 10^5	2.52 × 10^5

Abbreviations: K_SV, Stern-Volmer constant; K_q, bimolecular quenching rate constant.

Figure 6 WAXS patterns of PC membrane system in the absence (A) and presence (B) of AntiOxCIN₃ with some temperatures highlighted in red.

Figure 7 SAXS patterns of a BPL membrane model system in the presence of AntiOxCIN₃ before (red line) and after (blue line) the transition between lamellar phase (L_α) to inverted hexagonal phase (H_II).