Red Blood Cell-Based Delivery Systems for the Release of Hemoglobin-Derived Peptides with In Vitro Antitumor Activities

Abstract

Background/Objectives: This study aimed to develop liposomes derived from lipids obtained from red blood cell membranes for potential use in antitumor applications. Hemoglobin hydrolysates exhibiting peptides with known antitumor activities were encapsulated within these liposomes. Methods: The developed liposomal systems were characterized by their physicochemical properties, including size, surface charge, and encapsulation efficiency, and tested in vitro against 4T1 breast cancer cells and NIH3T3 fibroblasts. Results: Results indicated that the liposomes achieved effective encapsulation (88.9%), with nanometer-scale sizes (ranging from 140.7 nm for Blank-Liposomes to 658.3 nm for Pep-Liposomes) and stable colloidal properties. Conclusions: Although cytotoxicity was limited, the use of liposomes from endogenous components, such as red blood cells, demonstrates promise as a complementary approach in anticancer therapy.

Keywords: antitumor; cell mimetics; in vitro; liposome; mammalian cell; nanosystems; peptides; red blood cell.

Figure 1

MALDI-TOF mass spectrum of bovine hemoglobin extract acquired in linear positive mode at m/z range of 2000–20,000, showing monomers of α and β chain ions in their singly charged ([M+H]+ = 15,059/15,964) and doubly charged ([M+2H]+ = 7529/7978) forms.

Figure 2

Mass spectrum of bovine hemoglobin obtained by MALDI-TOF mass spectrometry in ISD mode, depicting α (top) and β (bottom) chains.

Figure 3

Mass spectrum obtained by MALDI-TOF/TOF operated in LIFT^TM mode of precursor ion at [M + H]+ = 1275.1 (identified as hemorphin), with y-series ions labeled, derived from bovine hemoglobin tryptic hydrolysate.

Figure 4

Viability of breast cancer cells (4T1, A) and non-tumoral murine fibroblasts (NIH/3T3, B) (MTT assay) after 24, 48, and 72 h of exposure to intact bovine hemoglobin. ANOVA: significant difference among groups, p < 0.05 (Tukey post hoc test). Statistically significant difference (p < 0.05) relative to 32 μg/mL (α).

Figure 5

Viability of breast cancer cells (4T1, A) and murine fibroblasts (NIH3T3, B) (MTT assay) after 24, 48, and 72 h of exposure to peptides resulting from hydrolysis of bovine hemoglobin by trypsin. ANOVA: significant difference among groups, p < 0.05 (Tukey post hoc test). Statistically significant difference (p < 0.05) relative to control: 0 μg/mL (*), 32 μg/mL (α), 64 μg/mL (β), 128 μg/mL (χ), 256 μg/mL (δ), 512 μg/mL (ε), 24 h ($), and 48 h (£).

Figure 6

The size distribution (hydrodynamic diameter—Dh) profiles of Blank-Liposomes and Pep-Liposomes. The larger size of Pep-Liposomes correlates with hemoglobin-derived peptides’ encapsulation, as described in Table 1. Measurements were performed at 25 °C using three independent replicates.

Figure 7

Evaluation of viability of 4T1 breast cancer cells by the MTT assay after 24 h of exposure to Blank-Liposomes and Pep-Liposomes (loaded with hemoglobin-derived peptides). Statistically significant difference (p < 0.05) relative to control: 0 μg/mL (*), 56 μg/mL (β), and Blank-Liposomes (&). ANOVA: significant difference among groups, p < 0.05 (Tukey post hoc test). Different symbols indicate statistically significant differences among groups.