Treatment with the urea immunoliposome significantly inhibited the proliferation of hemangioma vascular endothelial cells (HVECs) in a time- and dose-dependent manner
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. for approximately 6?months. persistently inhibited the proliferation of HVECs and is expected to be used in clinical hemangioma treatment. for approximately 6?months. The HVECs we isolated and cultured were recognized by HVEC factor VIII related antigen (VIII-R Ag) immunostaining and VEGFR2 circulation cytometry. For the immunostaining with VIII-R Ag, subcultured endothelial cells were inoculated on a cover glass and incubated for 10?min in 0.3% H2O2 diluted in methanol to reduce endogenous peroxidase activity. After blocking with normal goat serum, they were then incubated with anti-VIII-R Ag (1:50 dilution, ZSBIO, Beijing, China), followed by an incubation with secondary antibody conjugated with biotin (ZSBIO) and development with ABC (ZSBIO) and diaminobenzidine reagent (Boster, Wuhan, China). Digital images were obtained using a Leica Photo Microscope (Q550CW, Leica, Germany). For VEGFR circulation cytometry, subcultured endothelial cells were suspended in chilly culture medium at a density of 5??106 cells per milliliter. Forty microliters of cell suspension were mixed with anti-VEGFR2 and managed at 4C for 30?min. After washing, they were incubated with secondary antibody conjugated with fluorescein isothiocyanate (Boster) at 4C for 30?min. The cells were analyzed with an automated fluorescence-activated Acipimox cell counter (Elite, Beckman Acipimox Coulter), with which 1,000,000 events were counted. The complete quantity of cells expressing VEGFR2 per 1,000,000 events was calculated, and the percentage was derived. Influence of urea immunoliposome on human vascular endothelial cell morphology The HVECs were passaged in 96-well plates at a density of 4??103 cells per well. Twenty-four hours later, the urea immunoliposomes diluted with culture medium were added to each well at different final concentrations of 0, 0.002%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, or 40%. Four wells were seeded per concentration. Changes in HVEC morphology were observed under an inverted phase contrast microscope (IMT-2, Olympus, Japan) after 24?h, 48?h, and 72?h. Influence of urea immunoliposome on human vascular endothelial cell proliferation The HVECs were passaged in 96-well plates at a density of 2??103 cells per well. Twenty-four hours later, urea immunoliposomes diluted with culture medium were added to each well at the same final concentrations, pointed out previously. After 24, 48, and 72?hours, the proliferation of HVECs was measured by the MTT method (Sigma) according to the manufacturers protocols. The median influence dose (ID50) was calculated by linear regression analysis. The HVECs were passaged in 24-well plates at a density of 1 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 culture medium, at four wells per group. Subsequently, the number of HVECs in each group was counted every day for 8?days. A growth curve was generated and the population doubling time was calculated using the following formula: doubling time =?-?logrepresents the culture period during which the cell grows in the log phase; is usually the cell number at the end of the log phase. On the eighth day, the cell numbers of each group were used to calculate the inhibition rate: test. em P /em ? ?0.05 was considered statistically significant. Results Urea immunoliposome characteristics Both the urea liposome and urea immunoliposome created a milky white suspension on Acipimox gross examination, as shown in Physique?1A, which remained stable and did not obviously switch in appearance for 6?months at 4C. The urea immunoliposomes diluted 100 occasions showed a typical liposome morphology under a transmission electron microscope, as shown in Physique?1B. The urea immunoliposomes were spherical or near spherical, large unilamellar liposomes with a diameter of 150 to 200?nm. A nucleolar structure and lipid-coating structure were obvious. The encapsulation percentage of urea liposomes was 54.4%, according to Sephadex-50 column elution analysis. The coupling rate of anti-VEGFR to urea immunoliposomes was 36.84%. Open in a separate window Physique 1 Urea immunoliposome morphology. (A) Both the IGFBP6 urea liposome and urea immunoliposome generated a suspension that appeared milky white upon gross inspection. (B) Urea immunoliposomes diluted 100 occasions observed under a transmission electron microscope (150,000). Preparation and culture of hemangioma vascular endothelial cells The HVECs were isolated by tissue culture. Approximately 3?days after inoculation, the endothelial cells migrated quickly from.