[show abstract][hide abstract] ABSTRACT: Transcription factors (TFs) can direct cell fate by binding to DNA and regulating gene transcription. Controlling the intracellular levels of specific TFs can therefore enable reprogramming of cellular function and differentiation. Direct delivery of recombinant TFs to target cells can thus have widespread therapeutic value, but has remained challenging due to structural fragility of TFs and inefficient membrane transduction. Here we describe the functional delivery of TFs using degradable polymeric nanocapsules to drive cellular differentiation. The nanocapsules were synthesized with poly(ethylene) glycol (PEG)-based monomers and intracellularly-degradable crosslinkers. Physical properties and release kinetics of the nanocapsules were optimized through tuning of monomer and crosslinker ratios to achieve enhanced delivery of cargo destined for the nuclei. The nanocapsules did not display cytotoxicity in primary cell lines up to concentrations of 5 μm. A recombinant myogenic transcription factor, MyoD, was delivered to the nuclei of myoblast cells using degradable nanocapsules to induce myogenic differentiation. MyoD was confirmed to be delivered to the nuclei of myoblasts using confocal microscopy and was demonstrated to be active in transcription through a luciferase-based reporter assay. More importantly, delivered MyoD was able to drive myoblast differentiation as evidenced by the hallmark elongated and multinuclear morphology of myotubes. The activation of downstream cascade was also confirmed through immunostaining of late myogenic markers myogenin and My-HC. The efficiency of differentiation achieved via nanocapsule delivery is significantly higher than that of native MyoD, and is comparable to that of plasmid transfection. The encapsulated MyoD can also withstand prolonged protease treatment and remain functional. The ease of preparation, biocompatibility and effective cargo delivery make the polymeric nanocapsule a useful tool to deliver a variety of recombinant TFs for therapeutic uses.
[show abstract][hide abstract] ABSTRACT: Direct delivery of proteins to the cytosol of cells holds tremendous potential in biological and medical applications. Engineering vehicles for escorting proteins to the cytosol in a controlled release fashion has thus generated considerable interest. We report here the preparation of redox-responsive single-protein nanocapsules for intracellular protein delivery. Through in situ interfacial polymerization, the target protein is noncovalently encapsulated into a positively-charged polymeric shell interconnected by disulfide-containing crosslinkers. The dissociation of the polymeric shell under reducing conditions and the subsequent release of protein were confirmed using cell-free assays in the presence of glutathione (GSH). The nanocapsules were demonstrated to be efficiently internalized into the cells and to release the protein in the reducing cytosol. Using the nanocapsule as a vehicle, we showed that active caspase 3 (CP-3) can be delivered and can induce apoptosis in a variety of human cancer cell lines, including HeLa, MCF-7 and U-87 MG. Our approach therefore presents an effective intracellular protein delivery strategy for therapeutic, diagnostic and reprogramming applications.
[show abstract][hide abstract] ABSTRACT: Proteins play a crucial role in life, taking part in all vital processes in the body. In the past decade, there was increasing interest in delivering active forms of proteins to specific cells and organs. Intracellular protein delivery holds enormous promise for biological and medical applications, including cancer therapy, vaccination, regenerative medicine, treatment for loss-of-function genetic diseases and imaging. This tutorial review surveys recent developments in intracellular protein delivery using various nanocarriers. Methods such as lipid-mediated colloidal systems, polymeric nanocarriers, inorganic systems and protein-mediated carriers are reviewed. Advantages and limitations of current strategies, as well as future opportunities and challenges are also discussed.
Chemical Society Reviews 07/2011; 40(7):3638-55. · 24.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Proteins possess distinct intracellular roles allowing them to have vast therapeutic applications. However, due to poor cellular permeability and fragility of most proteins, intracellular delivery of native, active proteins is challenging. We describe a biomimetic protein delivery vehicle which is degradable upon the digestion by furin, a ubiquitous intracellular protease, to release encapsulated cargos. Proteins were encapsulated in a nanosized matrix prepared with monomers and a bisacrylated peptide cross-linker which can be specifically recognized and cleaved by furin. Release of encapsulated protein was confirmed in a cell-free system upon proteolytic degradation of nanocapsules. In vitro cell culture studies demonstrated successful intracellular delivery of both nuclear and cytosolic proteins and confirmed the importance of furin-degradable construction for native protein release. This endoprotease-mediated intracellular delivery system may be extended to effectively deliver various biological therapeutics.
[show abstract][hide abstract] ABSTRACT: We describe a FRET-based protease detection strategy, using a single-fluorescent-protein nanogel as donor and a dark quencher as acceptor linked by a photolabile caged-peptide. This design enables probing of protease activity in a UV-responsive fashion.
Chemical Communications 09/2010; 46(35):6467-9. · 6.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Target proteins can be functionally encapsulated using a cocoon-like polymeric nanocapsule formed by interfacial polymerization. The nanocapsule is cross-linked by peptides that can be proteolyzed by proteases upon which the protein cargo is released. The protease-mediated degradation process can be controlled in a spatiotemporal fashion through modification of the peptide cross-linker with photolabile moieties. We demonstrate the utility of this approach through the cytoplasmic delivery of the apoptosis inducing caspase-3 to cancer cells.