DNA and Polylysine Adsorption and Multilayer Construction onto Cationic Lipid-Coated Microbubbles

Abstract

We report on a novel application of the layer-by-layer (LbL) assembly technique to attach multiple layers of DNA and poly-l-lysine (PLL) onto preformed lipid-coated microbubbles to increase the DNA loading capacity. We first measured the effects of the cationic lipid fraction and salt concentration on the microbubble stability. Microbubble production and stability were robust up to a cationic lipid fraction of 40 mol % in 10 mM NaCl. DNA adsorption was heterogeneous over the microbubble shell and occurred primarily on the condensed phase domains. The amount of adsorbed DNA, and subsequently adsorbed PLL, increased linearly with the fraction of cationic lipid in the shell. DNA loading was further enhanced by the LbL assembly method to construct polyelectrolyte multilayers (PEMs) of DNA and PLL. PEM buildup was demonstrated by experimental results from zeta potential analysis, fluorescence microscopy, UV spectroscopy, and flow cytometry. The PEMs exhibited two growth stages and were heterogeneously distributed over the microbubble surface. The DNA loading capacity onto the microbubbles was enhanced by over 10-fold by using five paired layers. However, the PEM shell did not prevent oscillation or destruction during ultrasound insonification. These results suggest that the surface can be compartmentalized to make multifunctional, high-payload ultrasound contrast agents for targeted gene therapy.

Driving delivery vehicles with ultrasound

“…The lipid shell expands, buckles and compresses with each acoustic cycle, recoating as needed to cover bubble fragments [27][28][29][30][31][32]. A thick layer of DNA can be applied to the surface or, alternatively, liposomes or other nanoparticles can be attached to the membrane, each enhancing the payload [16,33,34]. Protein and polymer-shelled microbubbles have also been functionalized to carry targeting ligands [35] and genetic payloads [36,37].…”

Assessment of hepatic VX2 tumors with combined percutaneous transhepatic lymphosonography and contrast-enhanced ultrasonographic imaging

“…www.wjgnet.com At the same time, microbubbles are used not only for contrast enhancement of ultrasound images and improvement of diagnosis, but also for delivery of drugs and genes [36][37][38][39][40] . The ability to localize lymphatic vessels in tumors may be of value for a new route to the administration of drugs, gene and immunotherapy, etc.…”

Crucial factors and emerging concepts in ultrasound-triggered drug delivery

“…As an example, Frenkel et al [10] used albumin-shelled microbubbles as a template for pDNA attachment. A layer-by-layer approach can be used to alternately deposit cationic polymers and anionic nucleic acids on the microbubble shell, which clearly improves the electrostatic loading of the microbubbles with nucleic acids [11]. Sirsi et al [12] recently reported on lipid-shelled microbubbles that were covalently coated with PolyEthyleneGlycol-PolyEthyleneImine (PEG-PEI) copolymers, followed by the electrostatic deposition of pDNA onto such cationically charged microbubbles.…”