Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer.
ABSTRACT The direct intracellular delivery of proteins, or active peptide domains, has, until recently, been difficult to achieve due primarily to the bioavailability barrier of the plasma membrane, which effectively prevents the uptake of macromolecules by limiting their passive entry. Traditional approaches to modulate protein function have largely relied on the serendipitous discovery of specific drugs and small molecules which could be delivered easily into the cell. However, the usefulness of these pharmacological agents is limited by their tissue distribution and unlike 'information-rich' macromolecules, they often suffer from poor target specificity, unwanted side-effects, and toxicity. Likewise, the development of molecular techniques, over the past several decades, for gene delivery and expression of proteins has provided for tremendous advances in our understanding of cellular processes but has been of surprisingly little benefit for the management of genetic disorders. Apart from these gains however, the transfer of genetic material into eukaryotic cells either using viral vectors or by non-viral mechanisms such as microinjection, electroporation, or chemical transfection remains problematic. Moreover, in vivo, gene therapy approaches relying on adenoviral vectors are associated with significant difficulties relating to a lack of target specificity and toxicity which have contributed to poor performance in several clinical trials. Remarkably, the recent identification of a particular group of proteins with enhanced ability to cross the plasma membrane in a receptor-independent fashion has led to the discovery of a class of protein domains with cell membrane penetrating properties. The fusion of these protein transduction domain peptide sequences with heterologous proteins is sufficient to cause their rapid transduction into a variety of different cells in a rapid, concentration-dependent manner. Moreover, this novel technique for protein and peptide delivery appears to circumvent many problems associated with DNA and drug based methods. This technique may represent the next paradigm in our ability to modulate cell function and offers a unique avenue for the treatment of disease.
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ABSTRACT: PTD-fusion protein technology was used to transduce heat shock protein 27 (HSP27), an anti-apoptotic protein, into human lens epithelial cells (HLECs) (SRA01/04). The protein transduction domain (PTD) of the 11-amino acid YGRKKRRQRRR was tagged at the N-terminus of HSP27. The fusion protein was purified from bacteria transformed with a pKYB-PTD-HSP27 construct. The HLECs were incubated with PTD-HSP27-FITC and the fluorescence inside HLECs was found by fluorescence microscopic examination. To test the ability of PTD-HSP27 to pass through the corneas, PTD-HSP27-FITC was dropped onto the conjunctival sacs of rabbits; fluorescent labeled PTD-HSP27 was then observed in the rabbit aqueous humor. After being incubated with the PTD-HSP27 protein and irradiated with ultraviolet-B (UVB) light, HLECs was analyzed by flow cytometry, Hoechst 33258 staining and measurement of the potential of the mitochondrial transmembrane. HLECs incubated with PTD-HSP27 had a lower apoptotic rate and a higher mitochondrial membrane potential than the control cells. PTD-HSP27 appears to be sufficient to protect HLECs against UVB-induced apoptosis.Experimental Eye Research 01/2014; · 3.03 Impact Factor
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ABSTRACT: The clinical applications of protein drugs are restricted by absence of viable protein delivery vehicles. Here, we report on reduction and pH dual-sensitive crosslinked polymersomes based on poly(ethylene glycol)-poly(acrylic acid)-poly(2-(diethyl amino)ethyl methacrylate) (PEG-PAA-PDEA) triblock copolymer for efficient intracellular delivery of proteins and potent induction of cancer cell apoptosis. PEG-PAA-PDEA (1.9-0.8-8.2 kg/mol) was synthesized by controlled reversible addition-fragmentation chain transfer (RAFT) polymerization and further modified with cysteamine, to yield thiol-containing PEG-PAA(SH)-PDEA copolymer. PEG-PAA(SH)-PDEA was water soluble at acidic and physiological pH but formed robust and mono-disperse polymersomes with an average size of ca. 35 nm upon increasing pH to 7.8 or above followed by oxidative crosslinking. These disulfide-crosslinked polymersomes while exhibiting excellent colloidal stability were rapidly dissociated in response to 10 mM glutathione (GSH) at neutral or mildly acidic conditions. Notably, these polymersomes could efficiently load proteins like bovine serum albumin (BSA) and cytochrome C (CC). The in vitro release studies revealed that protein release was fast and nearly quantitative under the intracellular-mimicking reducing environment. Confocal microscopy observations showed that these dual-sensitive polymersomes efficiently released FITC-CC into MCF-7 cells in 6 h. Most remarkably, MTT assays showed that CC-loaded dual-sensitive polymersomes induced potent cancer cell apoptosis, in which markedly decreased cell viabilities of 11.3, 8.1 and 52.7% were observed for MCF-7, HeLa and 293T cells, respectively, at a CC dosage of 160 μg/mL. In contrast, free CC caused no cell death under otherwise the same conditions. These dual-bioresponsive polymersomes have appeared as a multifunctional platform for active intracellular protein release.Acta biomaterialia 01/2014; · 5.09 Impact Factor
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ABSTRACT: It is an attractive strategy to develop a recombinant bacterial vector vaccine by expressing exogenous protective antigen to induce the immune response, and the main concern is how to enhance the cellular internalization of antigen produced by bacterial vector. Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides which facilitate cellular uptake of various molecular cargoes and therefore have great potentials in vector vaccine design. In this work, eleven different CPPs were fused to the C-terminus of EGFP respectively, and the resultant EGFP-CPP fusion proteins were expressed and purified to assay their cross-membrane transport in macrophage J774 A.1 cells. Among the tested CPPs, TAT showed an excellent capability to deliver the cargo protein EGFP into cytoplasm. In order to establish an efficient antigen delivery system in Escherichia coli, the EGFP-TAT synthesis circuit was combined with an in vivo inducible lysis circuit PviuA-E in E. coli to form an integrated antigen delivery system, the resultant E. coli was proved to be able to lyse upon the induction of a mimic in vivo signal and thus release intracellular EGFP-TAT intensively, which were assumed to undergo a more efficient intracellular delivery by CPP to evoke protective immune responses. Based on the established antigen delivery system, the protective antigen gene flgD from an invasive intracellular fish pathogen Edwardsiella tarda EIB202, was applied to establish an E. coli recombinant vector vaccine. This E. coli vector vaccine presented superior immune protection (RPS = 63%) under the challenge with E. tarda EIB202, suggesting that the novel antigen delivery system had great potential in bacterial vector vaccine applications.Fish & Shellfish Immunology 04/2014; · 2.96 Impact Factor