Synthesis, acoustic stability, and pharmacologic activities of papaverine-loaded echogenic liposomes for ultrasound controlled drug delivery

Abstract

Background: development of encapsulated therapeutics that could be released upon ultrasound exposure has strong implications for enhancing drug effects at the target site. We have developed echogenic liposomes (ELIP) suitable for ultrasound imaging of blood flow and ultrasound-mediated intravascular drug release. Papaverine was chosen as the test drug because its clinical application requires high concentration in the target vascular bed but low concentration in the systemic circulation.

Methods: the procedure for preparation of standard ELIP was modified by including Papaverine hydrochloride in the lipid hydration solution, followed by three freeze-thaw cycles to increase encapsulation of the drug. Sizing and encapsulation pharmacokinetics were performed using a Coulter counter and a phosphodiesterase activity assay. Stability of Papaverine-loaded ELIP (PELIP) was monitored with a clinical diagnostic ultrasound scanner equipped with a linear array transducer at a center frequency of 4.5 MHz by assessing the mean digital intensity within a region of interest over time. The stability of PELIP was compared to those of standard ELIP and Optison.

Results: relative to standard ELIP, PELIP were larger (median diameter = 1.88 +/- 0.10 microm for PELIP vs 1.08 +/- 0.15 microm for ELIP) and had lower Mean Gray Scale Values (MGSV) (92 +/- 24.8 for PELIP compared to 142.3 +/- 10.7 for ELIP at lipid concentrations of 50 microg/ml). The maximum loading efficiency and mean encapsulated concentration were 24% +/- 7% and 2.1 +/- 0.7 mg/ml, respectively. Papaverine retained its phosphodiesterase inhibitory activity when associated with PELIP. Furthermore, a fraction of this activity remained latent until released by dissolution of liposomal membranes with detergent. The stability of both PELIP and standard ELIP were similar, but both are greater than that of Optison.

Conclusions: our results suggest that PELIP have desirable physical, biochemical, biological, and acoustic characteristics for potential in vivo administration and ultrasound-controlled drug delivery.

Figure 1

A. Effects of different papaverine stock solution concentration on the encapsulation efficiency of papaverine in echogenic liposomes. B. Effects of different papaverine stock solution concentration on encapsulation concentration of papaverine in echogenic liposomes.

Figure 2

Size distribution of standard echogenic liposomes and papaverine-loaded echogenic liposomes as measured by a Beckman-Coulter Multisizer 3 counter using a 20 μm aperture tube.

Figure 3

Phosphodiesterase inhibitory activities of papaverine-loaded echogenic liposomes with or without detergent treatment. The PDE-Glo^™ phosphodiesterase assay was performed in a 96-well plate using 0.67 mU of PDE from bovine brain, 0.0625 μM cAMP substrate, and the indicated amount of Papaverine. The PDE and papaverine were preincubated together for 5 min. The substrate was added, and the reactions were incubated for an additional 15 min at room temperature. The reaction was terminated with a buffer containing 100mM IBMX. Luminescence was developed with a detection buffer and Kinase Glo solution and subsequently read with a Tecan Safire2^™ plate reader (Mannedorf/Zurich, Switzerland). Means and standard deviations were derived from 4 samples at each papaverine concentration.

Figure 4

A. Papaverine-loaded echogenic liposomes (PELIP) and B. Standard ELIP in a vial imaged with a 20-MHz intravascular ultrasound imaging catheter. The bright objects within the vial represent liposomal clusters. The central artifact represents the imaging catheter.

Figure 5

Echogenicity of PELIP and ELIP expressed as mean gray scale values (MGSV) at different lipid concentrations imaged with a 20-MHz intravascular ultrasound imaging catheter.

Figure 6

Loss of echogenicity of PELIP, standard ELIP, and Optison^™ suspensions during intermittently scanned harmonic B-mode imaging (fc = 4.5 MHz) at MI = 0.04 (Pr = 0.12 MPa). MI = Mechanical Index, Pr = peak rarefractional pressure, fc = center frequency.