The direct delivery of functional proteins into the cell cytosol is a key issue for protein therapy, with many current strategies resulting in endosomal entrapment. Protein delivery to the cytosol is challenging due to the high molecular weight and the polarity of therapeutic proteins. Here we review strategies for the delivery of proteins into cells, including cell-penetrating peptides, virus-like particles, supercharged proteins, nanocarriers, polymers, and nanoparticle-stabilized nanocapsules. The advantages and disadvantages of these approaches including cytosolar delivery are compared and contrasted, with promising pathways forward identified.
Transcription mediated by hypoxia-inducible factor (HIF-1) contributes to tumor angiogenesis and metastasis but is also involved in activation of cell-death pathways and normal physiological processes. Given the complexity of HIF-1 signaling, it could be advantageous to target a subset of HIF-1 effectors rather than the entire pathway. We compare the genome-wide effects of three molecules that each interfere with the HIF-1-DNA interaction: a polyamide targeted to the hypoxia response element, small interfering RNA targeted to HIF-1alpha, and echinomycin, a DNA-binding natural product with a similar but less specific sequence preference than the polyamide. The polyamide affects a subset of hypoxia-induced genes consistent with its binding site preferences. For comparison, HIF-1alpha siRNA and echinomycin each affect the expression of nearly every gene induced by hypoxia. Remarkably, the total number of genes affected by either polyamide or HIF-1alpha siRNA over a range of thresholds is comparable. The data show that polyamides can be used to affect a subset of a pathway regulated by a transcription factor. In addition, this study offers a unique comparison of three complementary approaches towards exogenous control of endogenous gene expression.
Regulation of endogenous genes by DNA-binding polyamides requires effective nuclear localization. Previous work employing confocal microscopy to study uptake of fluorophore-labeled polyamides has demonstrated the difficulty of predicting a priori the nuclear uptake of a given polyamide. The data suggest that dye identity influences uptake sufficiently such that a dye-conjugate cannot be used as a proxy for unlabeled analogs. Polyamides capable of nuclear localization unaided by fluorescent dyes are desirable due to size and other limitations of fluorophores. Recently, a polyamide-fluorescein conjugate targeted to the hypoxia response element (HRE) was found to inhibit vascular endothelial growth factor (VEGF) expression in cultured HeLa cells. The current study uses inhibition of VEGF expression as a biological read-out for effective nuclear localization of HRE-targeted polyamides. We synthesized a focused library of non-fluorescent, HRE-targeted polyamides in which the C-terminus ‘tail’ has been systematically varied. Members of this library bind the HRE with affinities comparable or superior to that of the fluorescein-labeled analog. Although most library members demonstrate modest or no biological activity, two non-fluorescent polyamides are reported with activity rivaling that of the previously reported fluorescein-labeled polyamide. We also show evidence that promoter occupancy by HIF-1, the transcription factor that binds the HRE, is inhibited by HRE-targeted polyamides.
We report a convenient new technique for the labeling of filamentous phage capsid proteins. Previous reports have shown that phage coat protein residues can be modified, but the lack of chemically distinct amino acids in the coat protein sequences makes it difficult to attach high levels of synthetic molecules without altering the binding capabilities of the phage. To modify the phage with polymer chains, imaging groups, and other molecules, we have developed chemistry to convert the N-terminal amines of the ~4,200 coat proteins into ketone groups. These sites can then serve as chemospecific handles for the attachment of alkoxyamine groups through oxime formation. Specifically, we demonstrate the attachment of fluorophores and up to 3,000 molecules of 2 kD poly(ethylene glycol) (PEG2k) to each of the phage capsids without significantly affecting the binding of phage-displayed antibody fragments to EGFR and HER2 (two important epidermal growth factor receptors). We also demonstrate the utility of the modified phage for the characterization of breast cancer cells using multicolor fluorescence microscopy. Due to the widespread use of filamentous phage as display platforms for peptide and protein evolution, we envision that the ability to attach large numbers of synthetic functional groups to their coat proteins will be of significant value to the biological and materials communities.
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