Intracellular Delivery of Bioactive Molecules using Light-Addressable Nanocapsules

Abstract

This paper describes a method by which molecules that are impermeable to cells are encapsulated in dye-sensitized lipid nanocapsules for delivery into cells via endocytosis. Once inside the cells, the molecules are released from the lipid nanocapsules into the cytoplasm with a single nanosecond pulse from a laser in the far red (645nm). We demonstrate this method with the intracellular release of the second messenger IP 3 in CHO-M1 cells, and report that calcium responses from the cells changed from a sustained increase to a transient spike when the average number of IP 3 released is decreased below 50 molecules per nanocapsule. We also demonstrate the delivery of a 23 kDa AGT fusion protein into Ba/F3 cells to inhibit a key player BCR-ABL in the apoptotic pathway. We show that an average of ~ 8 molecules of the inhibitor is sufficient to induce apoptosis in the majority of Ba/F3 cells. KeywordsLipids; Dye-Sensitized; Nanocapsule; Intracellular; Photolysis To uncover the spatiotemporal dynamics of cellular function, we need techniques that allow us to perturb cells over space and time in a controlled fashion. The most common approach for the delivery of bioactive molecules to cells in a highly spatiotemporally-resolved fashion is the two-photon release of a chemically-caged compound. 1-5 Caged molecules, however, suffer from a number of drawbacks. The design and synthesis of a new caged compound can be tedious and the caging of large protein molecules can be difficult, if not impossible. For intracellular delivery, the caged molecules must be permeable to the cell; this requirement, in turn, makes it difficult to control the precise concentration of the caged molecules inside the cell because of their preferential partitioning into different cellular organelles and membranes. These drawbacks of caged compounds have prompted us to develop lightaddressable lipid nanocapsules as a general platform for caging a wide range of bioactive molecules. 6-12 Nanocapsules based on lipid vesicles represent an emerging class of physical cages,, some of which also have been used recently for intracellular release. [13][14][15][16][17][18] Our first nanocapsules were lipid vesicles which we manipulated with optical tweezers and photolyzed using a single nanosecond laser pulse in the UV. 6,8,10 This capability was sufficient for delivering molecules extracellularly in a cell-culture setting. Unfortunately, the lipid vesicles were fragile and could not be stored in solution for more than a few days. This * To whom correspondence should be addressed chiu@chem.washington.edu. Supporting Information

Ultrafast Near‐Infrared Light‐Triggered Intracellular Uncaging to Probe Cell Signaling

“…The binding event then causes calcium (Ca 2+ ) release from the endoplasmic reticulum into the cytosol . Cells were incubated with plasmonic liposomes containing 730 × 10 −6 m IP 3 . Both MDA‐MB‐231 cancer cells and primary culture of dorsal root ganglia (DRG) neurons were tested.…”

“…Upon near‐infrared light illumination, photosensitizer molecules produce singlet oxygen to oxidize vehicle structure and thus uncage the packaged biomolecules, or the payload. Among different vehicles, photosensitizer‐modified liposome is one of the most widely used vehicles for photochemically triggered uncaging . For instance, Kohane and co‐workers synthesized a new photosensitizer that when incorporated into liposomes, enabled light‐triggered lipid oxidation and tetrodotoxin uncaging to block the sciatic nerve .…”

Bivalent inhibitors of protein kinases

“…For example, the attachment of a HIV-TAT peptide to a potent JNK1 inhibitor by Stebbins and coworkers, allowed the authors to achieve JNK1 inhibition both in cultured cells and in glucose-intolerant mice (Stebbins et al 2011). Furthermore, we have shown that it is possible to photothermally release bivalent inhibitors that are encapsulated in dye-sensitized lipid nanocapsules (Gregersen et al 2010). Examples of this type point to the likelihood of increased use of bisubstrate and bivalent inhibitors in cellular studies.…”

“…Like the bisubstrate inhibitors described above, the first cellular experiments with SNAP-tag-based bivalent inhibitors involved the use of an intra-cellular delivery method (Gregersen et al 2010). Specifically, a fully assembled bivalent inhibitor of Abl was encapsulated in dye-sensitized lipid nanocapsules, which were taken up by BCR-Abl-dependent Ba/F3 cells through endocytosis.…”

Conversion of a Single Polypharmacological Agent into Selective Bivalent Inhibitors of Intracellular Kinase Activity

“…7–9 By selectively labeling SNAPtag fusion proteins, which display a specificity ligand from their N- or C-termini, with an ATP-competitive inhibitor, potent and selective bivalent inhibitors of several kinases have been generated. 10–13 Our previously described SNAPtag-based bivalent inhibitors relied on the use of genetically-encoded peptide ligands that had previously been characterized to be selective for a targeted site of interest in biochemical screens (referred to here as consensus-binding motifs ) as secondary specificity ligands. While this strategy led to impressive selectivity amongst closely related kinases, consensus-binding motifs often possess modest affinity and selectivity for their respective targets.…”