Albumin Submicron Particles with Entrapped Riboflavin-Fabrication and Characterization

Abstract

Although riboflavin (RF) belongs to the water-soluble vitamins of group B, its solubility is low. Therefore, the application of micro-formulations may help to overcome this limiting factor for the delivery of RF. In this study we immobilized RF in newly developed albumin submicron particles prepared using the Co-precipitation Crosslinking Dissolution technique (CCD-technique) of manganese chloride and sodium carbonate in the presence of human serum albumin (HSA) and RF. The resulting RF containing HSA particles (RF-HSA-MPs) showed a narrow size distribution in the range of 0.9 to 1 μm, uniform peanut-like morphology, and a zeta-potential of -15 mV. In vitro release studies represented biphasic release profiles of RF in a phosphate buffered saline (PBS) pH 7.4 and a cell culture medium (RPMI) 1640 medium over a prolonged period. Hemolysis, platelet activation, and phagocytosis assays revealed a good hemocompatibility of RF-HSA-MPs.

Keywords: CCD-technique; biopolymer; immobilization; riboflavin.

Figure 1

Scheme of the fabrication procedure for the submicron human serum albumin (HSA) particles containing riboflavin (RF), i.e., modified Co-precipitation Crosslinking Dissolution technique (CCD-technique).

Figure 2

AFM images of RF containing HSA particles (RF-HSA-MPs) (top) and HSA particles with 4 mL DMSO without RF (HSA-MPs) (bottom) in three dimensional (3D)-mode. The size of the particles was determined from the height profiles in horizontal and vertical directions. The values included in the images are representative examples.

Figure 3

Analysis of intrinsic fluorescence in HSA-MPs and RF-HSA-MPs. Confocal micrograph of HSA-MPs in (A1) fluorescence mode and (A2) transmission mode, respectively and confocal micrograph of RF-HSA-MPs in fluorescence mode (B1) and transmission mode (B2), respectively. Fluorescence emission intensity in 3D color map surface images of (A3) HSA-MPs and (B3) RF-HSA-MPs at an excitation wavelength of 480 nm and an emission wavelength of 535 nm.

Figure 4

(a) Release profiles of RF in the phosphate buffered saline (PBS) pH 7.4 (●) and in the cell culture medium (RPMI) 1640 medium (♦) at room temperature calculated for the remaining RF concentration entrapped in the RF-HSA-MPs. The RF concentrations were fitted using the Pappas equation m (t)/m (∞) = k₁tⁿ + k₂t²ⁿ. Values are expressed as mean ± SD (n = 3). (b) Fickian release fraction (F) of RF-HSA-MPs in PBS and in RPMI. The release of RF in RPMI is much stronger in the domination by the Fickian diffusion, due to the greater RF concentration gradient between RF in the RF-HSA-MPs and the bulk RPMI-phase.

Figure 4

(a) Release profiles of RF in the phosphate buffered saline (PBS) pH 7.4 (●) and in the cell culture medium (RPMI) 1640 medium (♦) at room temperature calculated for the remaining RF concentration entrapped in the RF-HSA-MPs. The RF concentrations were fitted using the Pappas equation m (t)/m (∞) = k₁tⁿ + k₂t²ⁿ. Values are expressed as mean ± SD (n = 3). (b) Fickian release fraction (F) of RF-HSA-MPs in PBS and in RPMI. The release of RF in RPMI is much stronger in the domination by the Fickian diffusion, due to the greater RF concentration gradient between RF in the RF-HSA-MPs and the bulk RPMI-phase.

Figure 5

Hemolytic activity induced by HSA-MPs and RF-HSA-MPs for 3 h at 37 °C, concentration ranging from 0.5%, 1%, and 2%. Water and PBS served as positive (100%) and negative (0%) control, respectively. Data are presented as the mean percentage ± SD (n = 3).

Figure 6

Phagocytosis assay showing (A) three groups of white blood cells identified by their forward scatter (FSC) and side scatter (SSC): granulocytes, monocytes, and lymphocytes. Histograms representing fluorescence intensity of (B) HSA-MPs and (C) RF-HSA-MPs in the PE-A channel. Phagocytosis of FITC-labeled opsonized E. coli in granulocytes (D) and monocytes (G) (grey area: negative controls incubated at 0 °C; black area: granulocytes and monocytes with phagocytosed FITC E. coli). Representative histograms of fluorescence intensity of monocytes and granulocytes after interacting with (E,H) HSA-MPs and (F,I) RF-HSA-MPs (grey area: negative controls incubated at 0 °C; black line: granulocytes and monocytes incubated at 37 °C MPs; k stands for kilo). Data are representative of n = 3 independent phagocytosis assays showing the same trends.

Figure 7

Platelet activation assay showing (A) representative dot plots in APC-A/FITC-A channel for HSA-MPs and RF-HSA-MPs (upper row); CD42b (APC-A)/CD62P (FITC-A) dot plots gated for platelets at rest and after stimulation with HSA-MPs and RF-HSA-MPs (second row); CD42b (APC-A)/CD62P (FITC-A) dot plots gated for platelets after stimulation with arachidonic acid, arachidonic acid and HSA-MPs, arachidonic acid and RF-HSA-MPs (third row); CD42b (APC-A)/CD62P (FITC-A) dot plots gated for platelets after stimulation with epinephrine, epinephrine and HSA-MPs, epinephrine and RF-HSA-MPs (lower row); (B) FACS analysis of MPs platelet activation measured by the determination of CD62p/CD42 co-expression. The presence of particles did not have an influence on platelet activation. The agonists (arachidonic acid, epinephrine, and collagen) induced platelet activation independent from the presence of particles. Data are representative of n = 4 independent platelet activations showing the same trends and are presented as mean ± SD (n = 4).