Destruction thresholds of echogenic liposomes with clinical diagnostic ultrasound

Echogenic liposomes (ELIP) are submicron-sized phospholipid vesicles that contain both gas and fluid. With antibody conjugation and drug incorporation, these liposomes can be used as novel targeted diagnostic and therapeutic ultrasound contrast agents. The utility of liposomes for contrast depends upon their stability in an acoustic field, whereas the use of liposomes for drug delivery requires the liberation of encapsulated gas and drug payload at the desired treatment site. The objective of this study was twofold: (1) to characterize the stability of liposome echogenicity after reconstitution and (2) to quantitate the acoustic destruction thresholds of liposomes as a function of peak rarefactional pressure (P,), pulse duration (PD) and pulse repetition frequency (PRF). The liposomes were insonified in an anechoic sample chamber using a Philips HDI 5000 diagnostic ultrasound scanner with a L12-5 linear array. Liposome stability was evaluated with 6.9-MHz fundamental and 4.5-MHz harmonic B-mode pulses at various P, at a fixed PRF. Liposome destruction thresholds were determined using 6.0-MHz Doppler pulses, by varying the PD with a fixed PRF of 1.25 kHz and by varying the PRF with a fixed PD of 3.33 fLs. Videos or freeze-captured images were acquired during each insonation experiment and analyzed for echogenicity in a fixed region of interest as a function of time. An initial increase in echogenicity was observed for fundamental and harmonic B-mode imaging pulses. The threshold for acoustically driven diffusion of gas out of the liposomes; using 6.0-MHz Doppler pulses was weakly dependent upon PRF and PD. The rapid fragmentation thresholds, however, were highly dependent upon PRF and PD. The quantification of acoustic destruction thresholds of ELIP is an important first step in their development as diagnostic and drug delivery agents.