Cholesterol Levels Affect the Performance of AuNPs-Decorated Thermo-Sensitive Liposomes as Nanocarriers for Controlled Doxorubicin Delivery

Abstract

Stimulus-responsive liposomes (L) for triggering drug release to the target site are particularly useful in cancer therapy. This research was focused on the evaluation of the effects of cholesterol levels in the performance of gold nanoparticles (AuNPs)-functionalized L for controlled doxorubicin (D) delivery. Their interfacial and morphological properties, drug release behavior against temperature changes and cytotoxic activity against breast and ovarian cancer cells were studied. Langmuir isotherms were performed to identify the most stable combination of lipid components. Two mole fractions of cholesterol (3.35 mol% and 40 mol%, L1 and L2 series, respectively) were evaluated. Thin-film hydration and transmembrane pH-gradient methods were used for preparing the L and for D loading, respectively. The cationic surface of L allowed the anchoring of negatively charged AuNPs by electrostatic interactions, even inducing a shift in the zeta potential of the L2 series. L exhibited nanometric sizes and spherical shape. The higher the proportion of cholesterol, the higher the drug loading. D was released in a controlled manner by diffusion-controlled mechanisms, and the proportions of cholesterol and temperature of release media influenced its release profiles. D-encapsulated L preserved its antiproliferative activity against cancer cells. The developed liposomal formulations exhibit promising properties for cancer treatment and potential for hyperthermia therapy.

Keywords: Langmuir monolayers; anchoring; anticancer activity; controlled drug release; gold nanoparticles; liposomal formulations; temperature-sensitive nanocarriers.

Figure 1

Solubility of doxorubicin (D) in different salt solutions as a function of pH.

Figure 2

SEM images of different doxorubicin salts: (A) sulfate salt at pH 4, (B) sulfate salt at pH 7, (C) citrate salt at pH 7, and (D) phosphate salt at pH 7 (A–C, scale bar: 5 µm; D, scale bar: 10 µm).

Figure 3

(A) Langmuir isotherms of monolayers of pure DPPC, DPPC:DDAB in a molar ratio 3:1, cholesterol and ternary monolayers of DPPC:DDAB 3:1 and cholesterol at some selected mole fractions of cholesterol (x_Chol). (B) 1/C_s vs. surface pressure plots for monolayers of pure DPPC, DPPC:DDAB in a molar ratio 3:1, cholesterol and ternary monolayers in panel (A).

Figure 4

(A) Area per molecule and (B) compression modulus (1/C_s) plotted vs. the cholesterol mole fraction at the constant pressures indicated in the figure for monolayers of DPPC:DDAB 3:1 and cholesterol. (C) Excess area per molecule (ΔA_exc), and (D) excess free energy (ΔG_exc) plotted vs. the cholesterol mole fraction at the constant pressures indicated in the figure for monolayers of DPPC:DDAB 3:1 and cholesterol.

Figure 5

FE-SEM images of (A) L1, (B) L2, (C) AuNPs-L1, (D) AuNPs-L2, (E) L1-D, (F) L2-D, (G) AuNPs-L1-D, (H) AuNPs-L2-D, and (I) AuNPs (scale bar: 200 nm). TEM images of (J) L2 (scale bar: 0.5 µm), (K) L2-D (scale bar: 1 µm), (L,M) AuNPs-L2-D (scale bar: 0.5 and 0.1 µm, respectively) and (N) AuNPs (scale bar: 0.1 µm). L1 and L2: liposomes with different proportions of cholesterol, 3.35 mol% y 40 mol%, respectively; AuNPs: gold nanoparticles (15 nm), and D: doxorubicin.

Figure 6

Effects of temperature, (A) 37 °C and (B) 42 °C, on in vitro release of doxorubicin (D) from both liposomes (L1 and L2, with different proportions of cholesterol, 3.35 mol% and 40 mol%, respectively) and gold nanoparticles (AuNPs)-functionalized liposomes toward Hepes buffer pH 7.4. In vitro D release profiles from the free drug are also shown.

Figure 7

Evaluation of the cytotoxic activity of free doxorubicin (D), drug unloaded liposomes, non-functionalized and functionalized with gold nanoparticles (L2 and AuNPs-L2, respectively) and D-loaded liposomes (L2-D and AuNPs-L2-D, respectively). MDA-MB-231 breast cancer cells and SK-OV-3 ovarian cancer cells were treated for 2 h (A,B), followed by a recovery period of 70 h in drug-free medium, or cells were continuously exposed to compounds for 72 h (C,D, respectively). Finally, cell viability was determined by the resazurin assay. The concentrations of the unloaded L represented in the graphs are those used to deliver the concentrations of D shown on the x-axis of the graphs. Data are reported as means ± standard error of mean (SEM) and were obtained from two independent experiments. L2 corresponds to the liposome series with 40 mol% of cholesterol.