(44c) Stable Hydrogel-Anchored Liposomes With Double Stranded DNA Linkers

Due to similarity of liposomes to cell membranes, they are valuable biocompatible nanostructures for handling biological molecules. They have various biomedical applications, and they can afford targeted delivery of therapeutic components, but they usually show insufficient stability, limiting their utility as drug delivery vehicles. Although several approaches have been used for enhancing liposome stability, most of them suffer multi-step processing and lack of biocompatibility.

To improve liposome stability, we have developed a biomimetic lipid-based nanostructure which has potential application in gene and drug delivery. In a self-assembly procedure, the lipid bilayer of a liposome was covalently anchored to a biocompatible poly(ethylene) glycol (PEG) hydrogel core using programmable double-stranded DNA (dsDNA) linkers. These dsDNA linkers are composed of two 15-mer complementary single-stranded DNAs (ssDNAs) modified with cholesterol and vinyl groups. Hybridized dsDNA linkers were added to the lipid mixture used for formation of liposomes, and the lipid mixture was dried. Dry lipid film was rehydrated with PEG-diacrylate (PEG-DA) monomer solution, and small unilamellar liposomes were formed by extrusion. We thermally controlled the formation of hydrogels inside liposomes while preventing bulk gel formation. During the liposome formation process, the DNA linkers spontaneously insert into the lipid bilayer via hydrophilic cholesterol and link the bilayer to the hydrogel by direct incorporation of the vinyl group in a free radical polymerization process.

Size exclusion chromatography (SEC) of intact and surfactant-treated liposomes confirmed the formation of nanogels anchored to the lipid membrane. Polymerized particles had the same column retention time regardless of whether or not they were treated with surfactant. This confirms the formation of liposome-templated nanogels which have same hydrodynamic radius as liposomes. Conjugation of dsDNAs to the nanogel surface was also confirmed by SEC of liposomes containing dye-labeled dsDNAs. Transmission electron microscopy (TEM) showed ~100 nm nanoparticles before and after removal of unanchored lipids confirming the formation of PEG nanogels in liposomes. Dynamic light scattering (DLS) also showed ~120 nm size nanoparticles corresponding to hydrogel-anchored liposomes. The size of surfactant-treated liposomes decreased by ~20 nm due to the removal of unanchored lipids and dsDNA molecules attached to the outer leaflet of the lipid bilayer.

A dsDNA sensitive dye, Hoechst 33342, verified the location of dsDNA linkers on the surface of nanogels. The change in quantum yield of this dye upon specifically binding dsDNA was used to show that DNA was accessible on the surface of nanogels from which the non-anchored lipids had been stripped away by surfactant.

A dye dequenching assay was used to demonstrate the vastly improved stability of hydrogel-anchored nanostructures versus both standard liposomes and liposomes containing a hydrogel core without anchoring. In this assay, a pair of well-known dye and quencher (ANTS/QSY) were co-encapsulated in liposomes incubated in 20% serum at 37°C. Hydrogel-anchored liposomes showed ~22% release over 184 h incubation in serum, while standard liposomes and hydrogel-liposomes with no dsDNA anchors showed faster release of dye with 100% release obtained within 184 h. Although hydogel-liposomes with no anchor showed slower dye release in first 90 h, their overall stability was not comparable with anchored liposomes.

The fabricated lipid-polymer nanostructures can provide robust drug delivery vehicles with advantages of sustained drug release and the potential for targeted delivery. We have deployed these particles in the intracellular delivery of a peptide with anticancer properties. SEC confirmed the successful and efficient encapsulation of fluorescently labeled 17-amino acid peptide within the PEG hydrogel core. We included a pH-sensitive lipid in the membrane to trigger release in and disruption of late-stage endosomes. Release kinetics studies of these pH-sensitive liposomes showed that liposomes are stable at neutral pH while rapidly releasing cargo at acid pH.  We traced intracellular delivery of the nanoparticles and their cargo by fluorescent confocal microscopy. In this pathway, cholesterol-dsDNAs arranged on surface of nanogels can facilitate the intracellular delivery of peptide-encapsulated nanogels by disrupting late-stage endosomes.

These stable nanostructures represent a unique platform for protected delivery of oligonocleotides and therapeutic compounds and allow formation of templated nanogels with programmable molecular recognition sites.