Encapsulation of Iron-Saturated Lactoferrin for Proteolysis Protection with Preserving Iron Coordination and Sustained Release

Abstract

Lactoferrin (Lf) is a globular glycoprotein found mainly in milk. It has a very high affinity for iron(III) ions, and its fully saturated form is called holoLf. The antimicrobial, antiviral, anticancer, and immunomodulatory properties of Lf have been studied extensively for the past two decades. However, to demonstrate therapeutic benefits, Lf has to be efficiently delivered to the intestinal tract in its structurally intact form. This work aimed to optimize the encapsulation of holoLf in a system based on the versatile Eudragit^® RS polymer to protect Lf against the proteolytic environment of the stomach. Microparticles (MPs) with entrapped holoLf were obtained with satisfactory entrapment efficiency (90-95%), high loading capacity (9.7%), and suitable morphology (spherical without cracks or pores). Detailed studies of the Lf release from the MPs under conditions that included simulated gastric or intestinal fluids, prepared according to the 10th edition of the European Pharmacopeia, showed that MPs partially protected holoLf against enzymatic digestion and ionic iron release. The preincubation of MPs loaded with holoLf under conditions simulating the stomach environment resulted in the release of 40% of Lf from the MPs. The protein released was saturated with iron ions at 33%, was structurally intact, and its iron scavenging properties were preserved.

Keywords: lactoferrin; microparticles; protein delivery; protein encapsulation.

Figure 1

SEM images for empty (A–C) and holoLf-loaded (E–G) microparticles prepared from Eudragit^® RS followed by lyophilization shown with different magnifications (right panel shows the crack- and pinhole-free MP surfaces). (D) The size distribution histogram for empty MPs (based on 100 diameter measurements for each of the five independent samples) and (H) for MPs loaded with holoLf (based on 100 diameter measurements for each of the three independent samples).

Figure 2

Lf release profile for lyophilized and freshly prepared MPs. MPs suspended in 10 times diluted PBS were placed under magnetic stirring at 300 rpm at room temperature. The released protein was quantified by the BCA protocol using the supernatant from the MPs. Insert: Release profile presented in the first 2 h.

Figure 3

Scheme of the studies on the release of Lf from MPs performed in simulated gastric and intestinal fluids. (1A,2A,3A) are chromatograms obtained for supernatants collected from the MPs suspended in GF + E, GF, and IF, respectively, after 1 h of incubation. (1B,2B) are chromatograms obtained for supernatants collected from the MPs kept for 1 h in GF + E and GF, respectively, followed by 23 h of incubation in IF. (3B) is a chromatogram for a supernatant collected from the MPs kept for 24 h in IF.

Figure 4

The Lf cumulative release under simulated gastric conditions. The percentage of the protein released from MPs was calculated as the ratio between the peak area of the protein in the HPLC separation and the peak area obtained for the sonicated sample in which the whole present protein was released. Incubation conditions: Lf(GF + E) MPs kept 1 h in GF + E followed by incubation in IF (red bars), Lf(GF) MPs kept 1 h in GF followed by incubation in IF, Lf(IF) MPs kept in IF. Lf (IF) bars with a dotted line present % of the protein released from the MPs in PBS taken from Figure 2 for comparison.

Figure 5

CD spectra of holoLf and apoLf as well as Lf released from MPs kept 1 h in GF + E followed by 23 h of incubation in IF–Lf(GF + E), kept 1 h in GF followed by 23 h of incubation in IF–Lf(GF), and kept 24 h in IF–Lf(IF). Spectra correspond to the sample analyzed using HPLC from Figure 3: chromatograms 1B, 2B, and 3B.

Figure 6

Calculated secondary structure composition in percentage (%) for PDB structure of diferric bovine Lf 1BLF [6], holoLf, apoLf, and Lf released from MPs kept 1 h in GF + E followed by 23 h of incubation in IF–Lf(GF + E), kept 1 h in GF followed by 23 h of incubation in IF–Lf(GF), and kept 24 h in IF–Lf(IF). Estimation was carried out using the BeStSel method [50]. Structure estimations correspond to the sample analyzed using HPLC from Figure 3: chromatograms 1B, 2B, and 3B.