Development and optimization of a doxorubicin loaded poly(lactic acid) contrast agent for ultrasound directed drug delivery

Abstract

An echogenic, intravenous drug delivery platform is proposed in which an encapsulated chemotherapeutic can travel to a desired location and drug delivery can be triggered using external, focused ultrasound at the area of interest. Three methods of loading poly(lactic acid) (PLA) shelled ultrasound contrast agents (UCA) with doxorubicin are presented. Effects on encapsulation efficiency, in vitro enhancement, stability, particle size, morphology and release during UCA rupture are compared by loading method and drug concentration. An agent containing doxorubicin within the shell was selected as an ideal candidate for future hepatocellular carcinoma studies. The agent achieved a maximal drug load of 6.2 mg Dox/g PLA with an encapsulation efficiency of 20.5%, showed a smooth surface morphology and tight size distribution (poly dispersity index=0.309) with a peak size of 1865 nm. Acoustically, the agent provided 19 dB of enhancement in vitro at a dosage of 10 microg/ml, with a half life of over 15 min. In vivo, the agent provided ultrasound enhancement of 13.4+/-1.6 dB within the ascending aorta of New Zealand rabbits at a dose of 0.15 ml/kg. While the drug-incorporated agent is thought to be well suited for future drug delivery experiments, this study has shown that agent properties can be tailored for specific applications based on choice of drug loading method.

Figure 1

Representation of the three drug loading methods explored: I:drug incorporation during fabrication (I-Dox-UCA); II: surface adsorption post fabrication (S-Dox-UCA); and III: surface adsorption during fabrication (H-Dox-UCA).

Figure 2

Final drug payload as a function of loading method and initial concentration (a), and corresponding encapsulation efficiency (b). H-Dox-UCA = , I-Dox-UCA = , S-Dox-UCA = . H-Dox-UCA approached a maximal drug load of 24.1 mg Dox/g PLA (encapsulation efficiency of 60.2%) at an initial loading concentration of 40.0 mg Dox/g PLA. Both the I-Dox-UCA and S-Dox-UCA samples reached peak drug payloads of 6.2 and 6.5 mg Dox/g PLA (encapsulation efficiencies of 20.5 and 21.9%) respectively at an initial loading concentration of 30.0 mg Dox/g PLA.

Figure 3

Images of drug loaded agents. a) SEM after fabrication (Mag. = 9000x, Size bar = 2 µm). b) SEM after sample ruptured by sonication (Mag. = 50000X, Size bar = 500 nm). c) Fluorescent confocal micrograph showing Dox within the agent’s shell (Mag=100X, Size bar= 5 µ, (only larger UCA are visible using fluorescent microscopy)). Agent shown is a PLA agent with 3% (g Dox/g PLA) loaded within the shell of the agent. Morphology, core, and shell thicknesses were consistent with all three loading methods and all drug payloads.

Figure 4

Effects of 3% Dox loading on back scattering enhancement (a) and stability (b) in vitro by each of the three drug loading methods (-■- unloaded UCA, -▲- H-Dox-UCA, -▼- I-Dox-UCA, -♦- S-Dox-UCA). A significant decrease in in vitro enhancement was seen in S-Dox-UCA relative to the unloaded control (* p=0.0062), while a significant decrease in stability was measured for I-Dox-UCA relative to the unloaded control (** p<0.0001).

Figure 5

Effects of loading method and insonation on drug release (-■- H-Dox-UCA, -▲- S-Dox-UCA, -▼- I-Dox-UCA, -□- H-Dox-UCA + US, -△- S-Dox-UCA + US, -▽- I-Dox-UCA + US,). H-Dox-UCA samples showed significantly more burst (*p<0.0001) relative to the S-Dox-UCA or I-Dox-UCA samples (78% vs. 27% of the total Dox within the UCA). No samples showed any significant release when insonated (p>0.05). Data shown is for 3% Dox loading, but consistent for all drug loading concentrations (n=3, error bars=SEAM).

Figure 6

Doppler US scans of a rat kidney (a) pre injection, and (b) 8 sec post injection of 0.1 ml/kg of I-Dox-UCA (3% Dox loading). The agent was clearly detectable in vivo and provides image enhancement for roughly 45 seconds.

Figure 7

In vivo dose response of 3% I-Dox-UCA in the ascending aorta of two New Zealand rabbits. The agent showed marked enhancement at all dosages, with a peak US enhancement of 13.4 dB at a dose of 0.15 ml/kg and lasting roughly 5 mins.