Iron Oxide Nanoparticles with Supramolecular Ureido-Pyrimidinone Coating for Antimicrobial Peptide Delivery

Abstract

Antimicrobial peptides (AMPs) can kill bacteria by disrupting their cytoplasmic membrane, which reduces the tendency of antibacterial resistance compared to conventional antibiotics. Their possible toxicity to human cells, however, limits their applicability. The combination of magnetically controlled drug delivery and supramolecular engineering can help to reduce the dosage of AMPs, control the delivery, and improve their cytocompatibility. Lasioglossin III (LL) is a natural AMP form bee venom that is highly antimicrobial. Here, superparamagnetic iron oxide nanoparticles (IONs) with a supramolecular ureido-pyrimidinone (UPy) coating were investigated as a drug carrier for LL for a controlled delivery to a specific target. Binding to IONs can improve the antimicrobial activity of the peptide. Different transmission electron microscopy (TEM) techniques showed that the particles have a crystalline iron oxide core with a UPy shell and UPy fibers. Cytocompatibility and internalization experiments were carried out with two different cell types, phagocytic and nonphagocytic cells. The drug carrier system showed good cytocompatibility (>70%) with human kidney cells (HK-2) and concentration-dependent toxicity to macrophagic cells (THP-1). The particles were internalized by both cell types, giving them the potential for effective delivery of AMPs into mammalian cells. By self-assembly, the UPy-coated nanoparticles can bind UPy-functionalized LL (UPy-LL) highly efficiently (99%), leading to a drug loading of 0.68 g g^-1. The binding of UPy-LL on the supramolecular nanoparticle system increased its antimicrobial activity against E. coli (MIC 3.53 µM to 1.77 µM) and improved its cytocompatible dosage for HK-2 cells from 5.40 µM to 10.6 µM. The system showed higher cytotoxicity (5.4 µM) to the macrophages. The high drug loading, efficient binding, enhanced antimicrobial behavior, and reduced cytotoxicity makes ION@UPy-NH₂ an interesting drug carrier for AMPs. The combination with superparamagnetic IONs allows potential magnetically controlled drug delivery and reduced drug amount of the system to address intracellular infections or improve cancer treatment.

Keywords: antimicrobial peptide; cytocompatibility; intracellular delivery; iron oxide nanoparticles; supramolecular system; ureido-pyrimidinone.

Figure 1

Schematic illustration of (a,b) the UPy unities (UPy-NH₂, UPy-LL) forming dimers, stacks, and fibers, (c) fiber formation, and (d) schematic illustration of agglomerated IONs with a UPy shell containing UPy fibers [27].

Figure 2

(a) HAADF-STEM micrographs of the same single ION@UPy-NH₂ indicating the crystalline NP core (green circle) and an overlapping smaller particle creating Moiré fringes (red outline); (b) DPC_x (A–C) and (c) DPC_y (B–D) images showing more clearly the NP core and the amorphous coating. (A–C) and (B–D) indicate different detector segments. The red arrow indicates the coating thickness.

Figure 3

(a) HAADF-STEM micrographs of ION@UPy-NH₂ agglomerates; (b) DPC_x (A–C) and (c) DPC_y (B–D) images; and (d) iDPC color display.

Figure 4

(a,b) Cryo-TEM micrographs of ION@UPy-NH₂, and (c) ION@UPy-NH₂@UPy-LL (35.3 µM UPy-LL were bound to 0.1 g L⁻¹ ION@UPy-NH₂). The fibers are marked with orange arrows.

Figure 5

Cytocompatibility examination of ION, ION@PGA, and ION@UPy-NH₂ by resazurin assay for (a) HK-2 cells and (b) THP-1 cells, and live/dead staining of (c) HK-2 and (d) THP-1 cells. The living cells were stained green, and the dead cells were stained red. Scale bars represent 200 µm.

Figure 6

Fluorescent micrographs of internalization of ION@UPy-NH₂ by HK-2 and THP-1 cells after 5 min, 120 min, 24 h, and 48 h. The ION@UPy-NH₂ were labeled with 0.025 g L⁻¹ UPy-Cy5 (red, Figure S1), and the nuclei (DAPI) and cell skeleton (Phalloidin Alexa 488) of the cells were stained blue and green, respectively. Scale bars represent 30 µm. All samples were examined under the same settings.

Figure 7

(a) Binding of UPy-LL to different concentrations of ION@UPy-NH₂ and (b) Nile red assay of ION@UPy-NH₂@UPy-LL with different UPy-LL concentrations. Growth studies with E. coli and (c) different concentrations of ION@UPy-NH₂ and (d) various amounts of UPy-LL and ION@UPy-NH₂@UPy-LL complexes.

Figure 8

Cytocompatibility examination by resazurin assay for UPy-LL and ION@UPy-NH₂-UPy-LL in (a) HK-2 cells and (b) THP-1 cells, and live/dead staining of (c) HK-2 and (d) THP-1 cells. The living cells are colored green and the dead cells red. Scale bars represent 200 µm.

Figure 8

Cytocompatibility examination by resazurin assay for UPy-LL and ION@UPy-NH₂-UPy-LL in (a) HK-2 cells and (b) THP-1 cells, and live/dead staining of (c) HK-2 and (d) THP-1 cells. The living cells are colored green and the dead cells red. Scale bars represent 200 µm.