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Cell-penetrating peptides (CPPs) are routinely used for the delivery of macromolecules into live human cells. To enter the cytosolic space of cells, CPPs typically permeabilize the membrane of endosomes. In turn, several approaches have been developed to increase the endosomal membrane permeation activity of CPPs so as to improve delivery efficiencies. The endocytic pathway is, however, important in maintaining cellular homeostasis, and understanding how endosomal permeation impacts cells is now critical to define the general utility of CPPs. Herein, we investigate how CPP-based delivery protocols affect the endocytic network. We detect that, in some cases, cell penetration induces the activation of Chmp1b, Galectin-3, and TFEB, which are components of endosomal repair, organelle clearance, and biogenesis pathways, respectively. We also detect that cellular delivery modulates endocytosis and endocytic proteolysis. Remarkably, a multimeric analogue of the prototypical CPP TAT permeabilizes endosomes efficiently without inducing membrane damage responses. These results challenge the notion that reagents that make endosomes leaky are generally toxic. Instead, our data indicates that it is possible to enter cells with minimal deleterious effects.

Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol. We establish successful delivery of peptides, DNA plasmids, and a single-chain variable fragment antibody. We also demonstrate that delivery works in hard-to-transfect mammalian cells and under conditions typically inhibitory to cell-penetrating peptides. Mechanistically, UNC7938 destabilizes the membrane of endosomes. This, in turn, enhances the endosome-leakage activity of cell-penetrating peptides and facilitates the endosomal escape of macromolecules initially internalized by mammalian cells via endocytosis. This combined selective membrane-destabilization represents a new chemical space for delivery tools and provides a novel solution to the problem of endosomal entrapment that often limits the effectiveness of reagent-based delivery approaches.

The pharmacological effectiveness of oligonucleotides has been hampered by their tendency to remain entrapped in endosomes, thus limiting their access to cytosolic or nuclear targets. We have previously reported a group of small molecules that enhance the effects of oligonucleotides by causing their release from endosomes. Here, we describe a second novel family of oligonucleotide enhancing compounds (OECs) that is chemically distinct from the compounds reported previously. We demonstrate that these molecules substantially augment the actions of splice switching oligonucleotides (SSOs) and antisense oligonucleotides (ASOs) in cell culture. We also find enhancement of SSO effects in a murine model. These new compounds act by increasing endosome permeability and causing partial release of entrapped oligonucleotides. While they also affect the permeability of lysosomes, they are clearly different from typical lysosomotropic agents. Current members of this compound family display a relatively narrow window between effective dose and toxic dose. Thus, further improvements are necessary before these agents can become suitable for therapeutic use.

We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nanometers diameter for the delivery of amphipathic drugs, represented by doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the alpha v/beta 3 integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 daltons were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the alpha v/beta 3 integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles, than in the case of untargeted control micelles, or free doxorubicin. Consequently the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug.

Cholesterol modified mono-, di-, and tetrameric oligonucleotides were synthesized and hybridized with antisense oligonucleotides to study their incorporation in cationic liposomes together with the influence of this dendrimeric delivery system on biological activity. Electrostatic interactions seem to play the most important role during complexation with cationic lipids. This oligonucleotide formulation gives a small but significant increase in the inhibition of P-glycoprotein expression in a cellular system.

Selective delivery of antisense or siRNA oligonucleotides to cells and tissues via receptor-mediated endocytosis is becoming an important approach for oligonucleotide-based pharmacology. In most cases receptor targeting has been attained using antibodies or peptide-type ligands. Thus there are few examples of delivering oligonucleotides using the plethora of small-molecule receptor-specific ligands that currently exist. In this report we describe a facile approach to the generation of mono- and multi-valent conjugates of oligonucleotides with small molecule ligands. Using the sigma receptor ligand anisamide as an example, we describe conversion of the ligand to a phosphoramidite and direct incorporation of this moiety into the oligonucleotide by solid phase DNA synthesis. We generated mono- and tri-valent conjugates of anisamide with a splice switching antisense oligonucleotide (SSO) and tested their ability to modify splicing of a reporter gene (luciferase) in tumor cells in culture. The tri-valent anisamide-SSO conjugate displayed enhanced cellular uptake and was markedly more effective than an unconjugated SSO or the mono-valent conjugate in modifying splicing of the reporter. Significant biological effects were attained in the sub-100 nM concentration range.