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.
"Thus, in the present study, a cellular model of AD was induced by the hyperphosphorylation of endogenous tau. Moreover, we utilized the PTD of the HIV Tat protein to enhance the delivery of Hsp27, thereby enhancing its protective effect (Wadia and Dowdy 2005; Stetler et al. 2010). Conjugation with specific peptide sequences, which are termed PTDs or cell-penetrating peptides, improves the delivery of a range of agents, including antisense oligonucleotides, plasmids, microbeads, and liposomes, which suggests that these peptide sequences may represent Fig. 3 Effect of Tat-Hsp27 on tau hyperphosphorylation. "
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is an age-related disorder that causes a loss of brain function. Hyperphosphorylation of tau and the subsequent formation of intracellular neurofibrillary tangles (NFTs) are implicated in the pathogenesis of AD. Hyperphosphorylated tau accumulates into insoluble paired helical filaments that aggregate into NFTs; therefore, regulation of tau phosphorylation represents an important treatment approach for AD. Heat shock protein 27 (Hsp27) plays a specific role in human neurodegenerative diseases; however, few studies have examined its therapeutic effect. In this study, we induced tau hyperphosphorylation using okadaic acid, which is a protein phosphatase inhibitor, and generated a fusion protein of Hsp27 and the protein transduction domain of the HIV Tat protein (Tat-Hsp27) to enhance the delivery of Hsp27. We treated Tat-Hsp27 to SH-SY5Y neuroblastoma cells for 2 h; the transduction level was proportional to the Tat-hsp27 concentration. Additionally, Tat-Hsp27 reduced the level of hyperphosphorylated tau and protected cells from apoptotic cell death caused by abnormal tau aggregates. These results reveal that Hsp27 represents a valuable protein therapeutic for AD.
"PTDs can be used in several ways. They can be introduced into protein by chemical conjugation method or alternatively, can be geneticallyfused to the protein cDNA and expressed in host mammalian cells via transfection or they can also be produced in bacteria even though the mechanism of transduction still remains unknown   . To date, various TAT fusion proteins have been investigated for the treatment of NDDs . "
[Show abstract][Hide abstract] ABSTRACT: Neurodegeneration is the progressive loss of structure or function of neurons leading to neuronal death, usually associated with ageing. Some of the common neurodegenerative disorders include Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and Huntington's disease. Due to recent advancements in high-throughput technologies in various disciplines such as genomics, epigenomics, metabolomics and proteomics, there has been a great demand for detection of specific macromolecules such as hormones, drug residues, miRNA, DNA, antibodies, peptides, proteins, pathogens and xenobiotics at nano-level concentrations for in-depth understanding of disease mechanisms as well as for the development of new therapeutic strategies. The present review focuses on the management of age-related neurodegenerative disorders using proteomics and nanotechnological approaches. In addition, this review also highlights the metabolism and disposition of nano-drugs and nano-enabled drug delivery in neurodegenerative disorders.
Current Drug Metabolism 12/2014; 16(5). DOI:10.2174/1389200216666141208153303 · 2.98 Impact Factor
"First, in order to cross cell membrane barriers efficiently and reach cytoplasmic and nucleic compartments, we fused P42 to the protein transduction domain of TAT from HIV. This 11aa Cell Penetrating Peptide (CPP) can be efficiently used as a vehicle to deliver fused peptides into living cells [26,27], via endocytosis . We first tested P42-TAT fusion peptide in HeLa cells for its abilities to transduce cells, and to rescue polyQ-hHtt aggregation. "
[Show abstract][Hide abstract] ABSTRACT: Background
In Huntington¿s disease (HD), the ratio between normal and mutant Huntingtin (polyQ-hHtt) is crucial in the onset and progression of the disease. As a result, addition of normal Htt was shown to improve polyQ-hHtt-induced defects. Therefore, we recently identified, within human Htt, a 23aa peptide (P42) that prevents aggregation and polyQ-hHtt-induced phenotypes in HD Drosophila model. In this report, we evaluated the therapeutic potential of P42 in a mammalian model of the disease, R6/2 mice.ResultsTo this end, we developed an original strategy for P42 delivery, combining the properties of the cell penetrating peptide TAT from HIV with a nanostructure-based drug delivery system (Aonys® technology), to form a water-in-oil microemulsion (referred to as NP42T) allowing non-invasive per mucosal buccal/rectal administration of P42. Using MALDI Imaging Mass Spectrometry, we verified the correct targeting of NP42T into the brain, after per mucosal administration. We then evaluated the effects of NP42T in R6/2 mice. We found that P42 (and/or derivatives) are delivered into the brain and target most of the cells, including the neurons of the striatum. Buccal/rectal daily administrations of NP42T microemulsion allowed a clear improvement of behavioural HD-associated defects (foot-clasping, rotarod and body weights), and of several histological markers (aggregation, astrogliosis or ventricular areas) recorded on brain sections.Conclusions
These data demonstrate that NP42T presents an unprecedented protective effect, and highlight a new therapeutic strategy for HD, associating an efficient peptide with a powerful delivery technology.
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