Liposomes surface conjugated with human hemoglobin target delivery to macrophages

Abstract

Current strategies to deliver therapeutic molecules to specific cell and tissue types rely on conjugation of antibodies and other targeting ligands directly to the therapeutic molecule itself or its carrier. This work describes a novel strategy to deliver therapeutic molecules into macrophages that takes advantage of the native hemoglobin (Hb) scavenging activity of plasma haptoglobin (Hp) and the subsequent uptake of the Hb-Hp complex into macrophages via CD163 receptor-mediated endocytosis. The drug delivery system described in this work consists of Hb decorated liposomes that can encapsulate any therapeutic molecule of interest, in this case the model fluorescent dye calcein was used in this study. The results of this study clearly demonstrate that this delivery system is specific towards macrophages and demonstrates the feasibility of using this approach in targeted drug delivery.

Figure 1

Chemical routes for conjugating DSPE-PEG(2000)-Maleimide to Hb. A, Thiolated Hb was synthesized by reacting the side chain of Hb surface lysine residues with 2-iminothiolane, in order to add free thiol groups to the surface of Hb. B, DSPE-PEG(2000)-Maleimide was conjugated to native cysteine residues on the surface of Hb or thiolated lysine residues on the surface of Hb through maleimide-thiol coupling chemistry.

Figure 2

SEC and UV-Visible spectrum of Hb-liposomes. A, SEC purification of Hb-liposomes and unreacted Hb, showing that a fraction of Hb co-eluted with the liposomes. The solid line, A₄₁₅ is due to the Soret band of Hb; while the dashed line, A₄₉₃ is due to the encapsulated calcein inside the Hb-liposomes. B, UV-Visible spectrum of Hb-liposomes, control-liposomes, and free Hb. The spectrum of Hb-liposomes is clearly a summation of the spectra from control-liposomes and free Hb. The solid line represents Hb-liposomes, while the dashed line represents control-liposome and the dotted line represents free Hb.

Figure 3

SDS-PAGE of Hb-liposomes, control-liposomes and Hb. Hb-liposomes exhibit three bands corresponding to unmodified individual α (15.2 kDa) and β (15.9 kDa) subunits (46% of loaded protein), α and β subunits coupled with one DSPE-PEG(2000)-Maleimide molecule (18.2 kDa and 18.9 kDa, respectively, 42% of loaded protein), and α and β monomers coupled with two DSPE-PEG(2000)-Maleimide molecules (21.2 kDa and 21.9 kDa, respectively, 12% of loaded protein).

Figure 4

DLS of Hb-liposomes and control-liposomes. A, DLS size distribution of the maleimide-liposomes before (dashed line) and after (solid line) reaction with Hb; B, DLS diameter results of Hb-liposomes, control-liposomes, and size increase caused by covalent modification of the liposome surface with Hb. The statistics are based on results from three different batches of liposomes.

Figure 5

Calcein release from Hb-liposomes and control liposomes. Both Hb-liposomes and control-liposomes possess low rates of drug release. The solid line represents Hb-liposomes, while the dashed line represents control-liposomes.

Figure 6

SPR of Hb-liposomes and control-liposomes.

Figure 7

Cell uptake experiments. Hb-liposomes are specifically internalized by macrophages, and not non-macrophage cells, while control-liposomes are unspecifically taken up by the cells. A, Fluorescent images of the indicated cell line treated with 1 mM of the indicated liposomes. The green signal represents calcein, while the blue signal represents cell nuclei. Bars represent 100 μm. B, Cell count results from the fluorescent images. The solid line represents Hb-liposomes, while the dashed line represents control-liposomes.