Design and characterization of targeted ultrasound microbubbles for diagnostic use.

Targeted ultrasound (US) contrast agents represent, because of their size (1 to 5 µm), a unique class of diagnostic imaging agents enabling true vascular imaging of conditions like inflammation and tumor angiogenesis. The objective of this study was to develop technology for preparing targeted microbubbles with binding and acoustic properties compatible with diagnostic use. Phosphatidylcholine (PC) was shown to represent the most favorable wall material. Various thiolated peptide binders were effectively conjugated to PC-based microbubbles containing maleimide functionalized lipids (95:5) without the need for biotin-streptavidin or antibody technology. By optimizing the technology, specific targeting of the inflammatory target E-selectin and the angiogenic target VEGFR2 in the presence of 100% serum was achieved. Increased phospholipid chain length from 18 carbons to 22 carbons improved the stability of the microbubbles during US exposure, without compromising binding or acoustic properties.

Biodistribution of pH-responsive liposomes for MRI and a novel approach to improve the pH-responsiveness.

The potential of pH-sensitive paramagnetic liposomes as a probe for monitoring acidic pH in tumours with magnetic resonance imaging has recently been demonstrated. If the blood retention time is prolonged, such liposomes can accumulate in tumour interstitium due to increased vascular permeability and interstitial retention. In the present study, biodistribution studies in healthy rats showed rapid clearance of the pH-sensitive system dipalmitoylphosphatidylethanolamine (DPPE)/dipalmitoylglycerosuccinate (DPSG) liposomal GdDTPA-BMA from the blood circulation with most of the Gd dose in the liver at 15 min post intravenous injection. Incorporation of 1.5 mol% polyethylene glycol (PEG) grafted DPPE (DPPE-PEG) in the above-mentioned formulation resulted in a significantly prolonged blood circulation time. However, the relaxometric pH-response of the DPPE/DPSG/DPPE-PEG system decreased as a function of mol% DPPE-PEG. Therefore, a compromise would be necessary between long blood residence time and a suitable pH-sensitivity of the liposomes. A possible approach to compensate for the reduced pH-sensitivity was investigated. Gadofosveset, a low-molecular weight Gd-chelate with high affinity for albumin, was encapsulated within DPPE/DPSG liposomes. This promising system showed in blood a markedly higher relaxometric response than the corresponding system with GdDTPA-BMA, due to release of gadofosveset at low pH and subsequent binding to albumin.

Tuning the MR properties of blood-stable pH-responsive paramagnetic liposomes.

Paramagnetic pH-responsive liposomes have recently been suggested as a promising approach for monitoring by magnetic resonance imaging (MRI) pH changes in tumours. In the present study, the effects of variations in bilayer composition on the relaxometric properties of diacylphosphatidylethanolamine (PE)/dipalmitoylglycerosuccinate (DPSG) liposomal GdDTPA-BMA were investigated both in buffer and blood. A factorial experimental design was used with the variables PE chain length and mol% DPSG. All the relaxometric profiles displayed a semi-sigmoidal shape with a minimum plateau at high pH (r1(min)) and a maximum at low pH (r1(max,E)). Relevant sigmoidal curve fit parameters were evaluated by partial least squares regression. Systematic variations in the relaxometric response (r1(max,E)-r1(min)) were shown for the liposomal systems both in buffer and blood. The pH value at which the r1 was 20% of r1(max,E) relative to r1(min), i.e. pH20, decreased significantly both in buffer and blood as a function of the mol% DPSG. This phenomenon could be understood by the increased surface charge density with increasing mol% DPSG and, hence, higher barrier against liposome aggregation with consequent leakage of contrast agent. Furthermore, the pH relaxometric profiles in blood were shifted laterally to higher, and likely more clinically relevant pH values than the corresponding profiles in buffer. The liposome formulations displayed minimal leakage of contrast agent after prolonged incubation in blood at physiological pH and retained their pH sensitivity after pre-incubation in blood.