Urea immunoliposome inhibits human vascular endothelial cell proliferation for hemangioma treatment

Abstract

Background: Urea injection has been used in hemangioma treatment as sclerotherapy. It shrinks vascular endothelial cells and induces degeneration, necrosis, and fibrosis. However, this treatment still has disadvantages, such as lacking targeting and difficulty in controlling the urea dosage. Thus, we designed a urea immunoliposome to improve the efficiency of treatment.

Methods: The urea liposome was prepared by reverse phase evaporation. Furthermore, the urea immunoliposome was generated by coupling the urea liposome with a vascular endothelial growth factor receptor (VEGFR) monoclonal antibody using the glutaraldehyde cross-linking method. The influence of the urea immunoliposome on cultured human hemangioma vascular endothelial cells was observed preliminarily.

Results: Urea immunoliposomes showed typical liposome morphology under a transmission electron microscope, with an encapsulation percentage of 54.4% and a coupling rate of 36.84% for anti-VEGFR. Treatment with the urea immunoliposome significantly inhibited the proliferation of hemangioma vascular endothelial cells (HVECs) in a time- and dose-dependent manner.

Conclusions: The urea immunoliposome that we developed distinctly and persistently inhibited the proliferation of HVECs and is expected to be used in clinical hemangioma treatment.

Figure 1

Urea immunoliposome morphology. (A) Both the urea liposome and urea immunoliposome generated a suspension that appeared milky white upon gross inspection. (B) Urea immunoliposomes diluted 100 times observed under a transmission electron microscope (×150,000).

Figure 2

Hemangioma vascular endothelial cell (HVEC) culture and identification. (A) The HVECs were derived from tissues resected during the surgery of children aged 59 days who had strawberry hemangioma on the abdominal surface. The tissues were cut into small pieces, inoculated into culture flasks, and maintained in culture medium. After 3 days, the endothelial cells migrated quickly from the tissue pieces. One week later, some of the endothelial cells became confluent (×100). (B) The HVECs were identified with human vascular endothelial cells factor VIII related antigen (VIII-R Ag) immunostaining (×200). (C) The HVECs were also analyzed with VEGFR2 flow cytometry. The percentage of HVECs in cells that we isolated and subcultured was 59.1%.

Figure 3

Urea immunoliposome induced inhibition of hemangioma vascular endothelial cell (HVEC) proliferation. HVECs were passaged in 24-well plates at a density of 1 × 10⁴ cells per well. Twenty-four hours later, the cells were divided into five groups: three experimental groups treated with 2.6% urea, 2.6% urea liposome, or 2.6% urea immunoliposome, and two control groups treated with the same volume of liposome or normal medium at four wells per group. The number of cultured HVECs was subsequently counted every day for 8 days. The data are representative of three independent experiments.