Microfluidic manufacture of rt-PA -loaded echogenic liposomes

Abstract

Echogenic liposomes (ELIP), loaded with recombinant tissue-type plasminogen activator (rt-PA) and microbubbles that act as cavitation nuclei, are under development for ultrasound-mediated thrombolysis. Conventional manufacturing techniques produce a polydisperse rt-PA-loaded ELIP population with only a small percentage of particles containing microbubbles. Further, a polydisperse population of rt-PA-loaded ELIP has a broadband frequency response with complex bubble dynamics when exposed to pulsed ultrasound. In this work, a microfluidic flow-focusing device was used to generate monodisperse rt-PA-loaded ELIP (μtELIP) loaded with a perfluorocarbon gas. The rt-PA associated with the μtELIP was encapsulated within the lipid shell as well as intercalated within the lipid shell. The μtELIP had a mean diameter of 5 μm, a resonance frequency of 2.2 MHz, and were found to be stable for at least 30 min in 0.5 % bovine serum albumin. Additionally, 35 % of μtELIP particles were estimated to contain microbubbles, an order of magnitude higher than that reported previously for batch-produced rt-PA-loaded ELIP. These findings emphasize the advantages offered by microfluidic techniques for improving the encapsulation efficiency of both rt-PA and perflurocarbon microbubbles within echogenic liposomes.

Keywords: Echogenic liposomes; Microfluidic flow-focusing; Recombinant tissue-type plasminogen activator; Stroke treatment; Ultrasound-mediated thrombolysis.

Fig. 1

a Microfluidic device design showing the inner drug channels, outer lipid channels, orifice, and the outlet reservoir (b) zoomed-in view of the extrusion orifice, and c the microfluidic device

Fig. 2

Microbubble formation downstream of the orifice showing the effect of increasing flow rates on particle sizes. The flow rates in images (a–c) are 40, 50, and 63.5 µL/min respectively. Scale bar: 50 µm

Fig. 3

Microscopic images of particles obtained using (a) a low lipid concentration (2 mg/mL), and b higher lipid concentration (10 mg/mL)

Fig. 4

Multisizer measurements showing number-weighted (left) and volume-weighted (right) µtELIP size distributions at (a–b) 10 min, c–d 16 min, and e–f 32 min

Fig. 5

Average US attenuation of µtELIP suspended in 0.5 % BSA at 22.5 ± 0.5 °C measured after 30 min (N = 3). Error bars represent standard deviations

Fig. 6

Measured attenuation coefficients as a function of frequency (dashed lines) and theoretical fits (solid lines) based on the estimated shell parameters of the µtELIP. The gray band denotes the 95 % confidence interval for the fit

Fig. 7

a µtELIP diluted in 0.9 % saline without cobalt chloride quenching demonstrating the presence of adsorbed rt-PA on the µtELIP exterior surface (b) Fluorescent staining of a µtELIP showing the lipid layer in red and the calcein associated with encapsulated rt-PA in green without cobalt chloride (c) and after addition of cobalt chloride to quench fluorescence from rt-PA on the exterior of the liposomes

Fig. 8

The amount of rt-PA associated with the rt-PA and lipid mixture extruded through the microfluidic device (148.70 ± 30.80 µg/mL) compared with that associated with the µtELIP shell (64.30 ± 3.80 µg/mL)