Introduction.
Bevacizumab (BEV) is a humanized monoclonal antibody to vascular endothelial growth factor (VEGF) that can ameliorate atheroma progression, the mechanism of which is believed to be through inhibition of neovascularization. Systemically, BEV has multiple adverse effects, which may be diminished in a controlled release delivery vehicle.
Hypothesis.
The purpose of this study was to test the hypothesis that BEV release from echogenic liposomes (BEV-ELIP) could be enhanced by color Doppler ultrasound (US).
Methods.
BEV-ELIP were prepared by a rehydration/loading-freeze/thaw-centrifugation-lyophilization method. BEV-ELIP samples were resuspended in 50ml of porcine plasma and divided into two portions, one of which was subjected to 6 MHz color Doppler ultrasound (MI = 0.4) for 5 minutes. After various time intervals, aliquots were fractionated by centrifugation and assayed for immunoreactive BEV with a direct enzyme-linked immunosorbent assay (ELISA). We considered immunoreactive BEV in washed pellet suspensions to represent ELIP surface-exposed VEGF-binding sites. To assure that BEV was accurately measured in plasma, we repeated the release protocol with fluoresceinated BEV (FITC-BEV) loaded into ELIP by the same method; BEV was measured by quantitation of fluorescence emission using a SpectraMax M5 microplate reader.
Results.
BEV-ELIP encapsulation efficiency was 43.2 ± 9.7% (SD, n = 5); 34.1 ± 29.3% of the encapsulated BEV was spontaneously released within the first 30 minutes. US caused an additional 4.1% of BEV to be released from the surface exposed compartment. Fluorescence measurements indicated that 43.2 ± 6.2% (n = 3) of encapsulated FITC-BEV-ELIP was spontaneously released in 60 minutes and that US caused an additional 5.7% to be released in that period, but, overall, US caused an additional 100 μg of BEV to be released or exposed per aliquot.
Conclusions.
We have demonstrated that BEV-ELIP retains its VEGF-binding activity in a liposomal formulation and that clinical Doppler US can significantly increase that activity, both by releasing free BEV and by enhancing the surface exposure of the immunoreactive antibody. These results warrant investigation of the formulation’s anti-angiogenic efficacy in vitro and in vivo.