A Novel Artificial Hemoglobin Carrier Based on Heulandite-Calcium Mesoporous Aluminosilicate Particles

Abstract

Tetraethyl-orthosilicate (TEOS)-based nanoparticles are most extensively used as a silica-based hemoglobin carrier system. However, TEOS-based nanoparticles induce adverse effects on the hemoglobin structure. Therefore, a heulandite-calcium-based carrier was investigated as a novel silica-based hemoglobin carrier system. The heulandite-calcium mesoporous aluminosilicate particles (MSPs) were fabricated by a patented tribo-mechanical activation process, according to the manufacturer, and its structure was assessed by X-ray diffraction analysis. Upon hemoglobin encapsulation, alternation in the secondary and tertiary structure was observed. The hemoglobin-particle interactions do not cause heme degradation or decreased activity. Once encapsulated inside the particle pores, the hemoglobin shows increased thermal stability, and higher loading capacity per gram of particles (by a factor of >1.4) when compared to TEOS-based nanoparticles. Futhermore, we introduced a PEGlyted lipid bilayer which significantly decreases the premature hemoglobin release and increases the colloidal stability. The newly developed hemoglobin carrier shows no cytotoxicity to human umbilical vein endothelial cells (HUVEC).

Keywords: encapsulation; hemoglobin carrier; mesoporous; silica particles; spectroscopy.

Figure 1

Part I: (A) A representative transmission electron microscopy (TEM) image of MSPs (Heulandite-Calcium Mesoporous Aluminosilicate Particles). (B) Representative scanning electron microscopy (SEM) image of MSPs. (C) X-ray diffraction pattern of the MSPs. (D,E) Representative TEM and SEM images of empty liposomes, retrospectively. (F,G) Representative TEM and SEM images of LB-MSPs (lipid bilayer coated Heulandite-Calcium Mesoporous Aluminosilicate Particles) (white arrow: showing the lipid layers around the particle). Scale bar for all images, 100 nm. Part II: (H) Representative confocal images of a DOPE-LR labeled liposome (yellow color), a FITC-labeled Hb-MSP (purple color), and Hb-loaded LB-MSPs (purple color with yellow outside layer). Scale bar, 2 µm. Inserts: Representative confocal images of DOPE-LR labeled liposome, FITC-labeled Hb-MSP and Hb-loaded LB-MSPs. Scale bar, 1 µm.

Figure 1

Part I: (A) A representative transmission electron microscopy (TEM) image of MSPs (Heulandite-Calcium Mesoporous Aluminosilicate Particles). (B) Representative scanning electron microscopy (SEM) image of MSPs. (C) X-ray diffraction pattern of the MSPs. (D,E) Representative TEM and SEM images of empty liposomes, retrospectively. (F,G) Representative TEM and SEM images of LB-MSPs (lipid bilayer coated Heulandite-Calcium Mesoporous Aluminosilicate Particles) (white arrow: showing the lipid layers around the particle). Scale bar for all images, 100 nm. Part II: (H) Representative confocal images of a DOPE-LR labeled liposome (yellow color), a FITC-labeled Hb-MSP (purple color), and Hb-loaded LB-MSPs (purple color with yellow outside layer). Scale bar, 2 µm. Inserts: Representative confocal images of DOPE-LR labeled liposome, FITC-labeled Hb-MSP and Hb-loaded LB-MSPs. Scale bar, 1 µm.

Figure 2

(A) Nitrogen adsorption-desorption isotherms of MSPs and Hb-MSPs. (B) Plots of pore diameter vs. pore volume, as calculated from the desorption isotherms using the Barrett–Joyner–Helenda model.

Figure 3

(A) Thermogravimetric analysis curves of Hb-loaded MSPs with different initial Hb concentrations (0, 0.25, 0.5, 1, 1.5, 2, 3, 4 mg/mL; top to bottom). (B) Corresponding loading capacities of Hb into MSPs calculated by thermogravimetric analysis. (C) ABTS catalyzed by native Hb and Hb-loaded MSPs. The enzymatic activity of Hb was measured at 418 nm as the catalytic conversion of oxidation of ABTS.

Figure 4

Iron release from hemoglobin in the presence of increasing concentrations of MSPs. Data are means ±SD of two independent experiments. * p-value < 0.05, ** p-value < 0.01 vs. control preforming one way analysis of variance (ANOVA) by Fisher’s comparison test.

Figure 5

(A) Steady-state fluorescence spectra of Hb and Hb-MSPs at 278 nm excitation wavelength. (B) Heme degradation study of Hb-MSPs at 321 nm excitation wavelength.

Figure 6

Far−UV (A) and near-UV (B) circular dichroism spectra of Hb and Hb−MSPs at different MSP concentrations (25, 50, 100, 150, 200 µg/mL; bottom to top) and at Hb concentrations of 0.1 mg/mL (A) and 1 mg/mL (B).

Figure 7

(A,C) Fourier transform infrared spectra of heulandite−Ca MSPs (A,C), and free Hb and Hb−loaded MSPs (A), and empty liposomes and liposome−encapsulated MSPs (C). (B) Curve−fitting of the inverted second−derivate spectra of the amide I region of Hb and Hb−loaded MSPs.

Figure 8

(A) A typical differential scanning calorimetry (DSC) thermal denaturation profile for free Hb and Hb−loaded MSPs. Insets (a, b): raw DSC spectra for free Hb (a) and Hb−loaded MSPs (b). (B) DSC thermograms of empty liposomes and lipid−bilayer-coated MSPs. Insets (a, b): raw DSC spectrum for empty liposomes (a) and liposome−encapsulated MSPs (b). Heating rate, +10 °C/min.

Figure 9

Changes in anisotropy of the membrane probes (TMA−DPH and DPH).

Figure 10

(A) Dynamic light scattering for the mean hydrodynamic diameters of empty liposomes, MSPs, and liposome−encapsulated MSPs (LB−MSPs) (in 1 mM phosphate buffer, pH 7.4). (B) Zeta potentials before and after MSP encapsulation in the liposomes. The error bars represent the zeta potential data from the streaming potential measurements. (C) Release profiles of Hb−loaded MSPs and LB−Hb−MSPs (in phosphate−buffered saline, 37 °C, pH 7.4). The error bars represent the standard deviation.

Figure 11

Cell viability after 24 h exposure to increasing concentrations of Hb, Hb−loaded MSPs (Hb−MSPs), and liposome-encapsulated MSPs (LB−MSPs). No significant differences were seen vs. control preforming one way analysis of variance (ANOVA) by Fisher’s comparison test.