Magnetoliposomes Based on Shape Anisotropic Calcium/Magnesium Ferrite Nanoparticles as Nanocarriers for Doxorubicin

Abstract

Multifunctional lipid nanocarriers are a promising therapeutic approach for controlled drug release in cancer therapy. Combining the widely used liposome structure with magnetic nanoparticles in magnetoliposomes allies, the advantages of using liposomes include the possibility to magnetically guide, selectively accumulate, and magnetically control the release of drugs on target. The effectiveness of these nanosystems is intrinsically related to the individual characteristics of the two main components-lipid formulation and magnetic nanoparticles-and their physicochemical combination. Herein, shape-anisotropic calcium-substituted magnesium ferrite nanoparticles (Ca_0.25Mg_0.75Fe₂O₄) were prepared for the first time, improving the magnetic properties of spherical counterparts. The nanoparticles revealed a superparamagnetic behavior, high saturation magnetization (50.07 emu/g at 300 K), and a large heating capacity. Furthermore, a new method for the synthesis of solid magnetoliposomes (SMLs) was developed to enhance their magnetic response. The manufacturing technicalities were optimized with different lipid compositions (DPPC, DPPC/Ch, and DPPC/DSPE-PEG) originating nanosystems with optimal sizes for biomedical applications (around or below 150 nm) and low polydispersity index. The high encapsulation efficiency of doxorubicin in these magnetoliposomes was proven, as well as the ability of the drug-loaded nanosystems to interact with cell membrane models and release DOX by fusion. SMLs revealed to reduce doxorubicin interaction with human serum albumin, contributing to a prolonged bioavailability of the drug upon systemic administration. Finally, the drug release kinetic assays revealed a preferable DOX release at hyperthermia temperatures (42 °C) and acidic conditions (pH = 5.5), indicating them as promising controlled release nanocarriers by either internal (pH) and external (alternate magnetic field) stimuli in cancer therapy.

Keywords: doxorubicin; magnetic hyperthermia; magnetic nanoparticles; magnetoliposomes; mixed ferrites; shape-anisotropy.

Figure 1

Schematic representation of the synthesis methodology of DOX-loaded solid magnetoliposomes.

Figure 2

XRD pattern of the Ca_0.25Mg_0.75Fe₂O₄ nanoparticles and corresponding Rietveld analysis.

Figure 3

(A–C): High-Resolution Transmission Electron Microscopy (HR-TEM) images of Ca_0.25Mg_0.75Fe₂O₄ ferrite nanoparticles at different magnifications; (D): Aspect ratio distribution histogram; (E): Size histogram and distribution fit (R² = 0.96) and (F): Small Area Electron Diffraction (SAED) image with indexed diffraction planes.

Figure 3

(A–C): High-Resolution Transmission Electron Microscopy (HR-TEM) images of Ca_0.25Mg_0.75Fe₂O₄ ferrite nanoparticles at different magnifications; (D): Aspect ratio distribution histogram; (E): Size histogram and distribution fit (R² = 0.96) and (F): Small Area Electron Diffraction (SAED) image with indexed diffraction planes.

Figure 4

Tauc plot for shape-anisotropic Ca_0.25Mg_0.75Fe₂O₄ ferrite nanoparticles (the red line corresponds to the linear fit).

Figure 5

Magnetization hysteresis cycle of Ca_0.25Mg_0.75Fe₂O₄ ferrite nanoparticles at 5 K and 300 K. Inset: enlargement of the hysteresis loop in the low field region.

Figure 6

Magnetically-induced thermal response curve (temperature variation vs. time) of Ca_0.25Mg_0.75Fe₂O₄ nanoparticles (NPs) and SMLs for magnetic hyperthermia. One mg of nanoparticles were dispersed in 1 mL of water and subjected to an AMF with different field frequencies and strengths, over 30 min (1800 s).

Figure 7

(A) Molecular structure of doxorubicin (C₂₇H₂₉NO₁₁); (B) Absorption spectra of doxorubicin solutions, at 1 × 10⁻⁵ M, in several solvents; (C) Normalized fluorescence (at peak of maximum emission) spectra of doxorubicin (1 × 10⁻⁶ M, λ_exc = 480 nm) in several solvents.

Figure 8

Fluorescence spectra (λ_exc = 470 nm) of SMLs with a DPPC bilayer labeled with only NBD-C₁₂-HPC, with only Rh-DOPE and labeled with both NBD-C₁₂-HPC and Rh-DOPE.

Figure 9

Hydrodynamic diameter and polydispersity (PDI) evolution of an aqueous solution of DPPC-based SMLs during storage at 4 °C for 30 days.

Figure 10

(A): HR-TEM image of a solid magnetoliposome based on Ca_0.25Mg_0.75Fe₂O₄ ferrite nanoparticles. (B): Schematic representation of a SML, characterized by the presence of a core (cluster of magnetic nanoparticles) covered with a lipid bilayer.

Figure 11

Fluorescence spectra (λ_exc = 480 nm) of doxorubicin (2 × 10⁻⁶ M) in liposomes (without magnetic nanoparticles) and in SMLs containing Ca_0.25Mg_0.75Fe₂O₄ magnetic nanoparticles.

Figure 12

Fluorescence spectra (λ_exc = 480 nm) of doxorubicin in SMLs containing Ca_0.25Mg_0.75Fe₂O₄, before and after interaction with GUVs at 25 °C and 55 °C, for the different lipid formulations. (A) DPPC; (B) DPPC:DSPE-PEG; (C) DPPC:Ch.

Figure 12

Fluorescence spectra (λ_exc = 480 nm) of doxorubicin in SMLs containing Ca_0.25Mg_0.75Fe₂O₄, before and after interaction with GUVs at 25 °C and 55 °C, for the different lipid formulations. (A) DPPC; (B) DPPC:DSPE-PEG; (C) DPPC:Ch.

Figure 13

HSA fluorescence quenching (%) as a function of increasing doxorubicin concentration in free form and loaded in DPPC/DSPE-PEG-based liposomes. A nonlinear fit according to Equation (8) is presented (red lines).

Figure 14

In vitro kinetic release profile of doxorubicin encapsulated in SMLs at different conditions of temperatures and pH, with the triplicate mean fitted to the Weibull kinetic model. (A) DPPC SMLs; (B) DPPC:DSPE-PEG SMLs.

Figure 15

Size variation of DOX-loaded SMLs with increasing temperature at different pH values. (A) DPPC SMLs; (B) DPPC/DSPE-PEG SMLs. Mean size values and standard deviation are from triplicate assays.