| 1. |
- Nebogatova, Jekaterina, et al.
(författare)
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A Method for Using Cell-Penetrating Peptides for Loading Plasmid DNA into Secreted Extracellular Vesicles
- 2023
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Ingår i: Biomolecules. - 2218-273X. ; 13:12
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Tidskriftsartikel (refereegranskat)abstract
- The low bioavailability and high toxicity of plasmid DNA (pDNA)-based therapeutics pose challenges for their in vivo application. Extracellular vesicles (EVs) have great potential to overcome these limitations, as they are biocompatible native cargo carriers. Various methods for loading pDNA into EVs, including electroporation, sonication, and co-incubation, have been previously investigated, but their success has been questionable. In this study, we report a unique method for loading EVs with pDNA through transient transfection using cell-penetrating peptides (CPPs). With this method, we found a 104-fold increase in the expression levels of the luciferase reporter protein in recipient cells compared to the untreated cells. These data point to the high transfection efficacy and bioavailability of the delivered encapsulated nucleic acid. Furthermore, the in vivo experimental data indicate that the use of pDNA-loaded EVs as native delivery vehicles reduces the toxic effects associated with traditional nucleic acid (NA) delivery and treatment.
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| 2. |
- Porosk, Ly, et al.
(författare)
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Aggregation Limiting Cell-Penetrating Peptides Derived from Protein Signal Sequences
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24:5
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer’s disease (AD) is the most common neurodegenerative disease (ND) and the leading cause of dementia. It is characterized by non-linear, genetic-driven pathophysiological dynamics with high heterogeneity in the biological alterations and the causes of the disease. One of the hallmarks of the AD is the progression of plaques of aggregated amyloid-β (Aβ) or neurofibrillary tangles of Tau. Currently there is no efficient treatment for the AD. Nevertheless, several breakthroughs in revealing the mechanisms behind progression of the AD have led to the discovery of possible therapeutic targets. Some of these include the reduction in inflammation in the brain, and, although highly debated, limiting of the aggregation of the Aβ. In this work we show that similarly to the Neural cell adhesion molecule 1 (NCAM1) signal sequence, other Aβ interacting protein sequences, especially derived from Transthyretin, can be used successfully to reduce or target the amyloid aggregation/aggregates in vitro. The modified signal peptides with cell-penetrating properties reduce the Aβ aggregation and are predicted to have anti-inflammatory properties. Furthermore, we show that by expressing the Aβ-EGFP fusion protein, we can efficiently assess the potential for reduction in aggregation, and the CPP properties of peptides in mammalian cells.
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| 3. |
- Porosk, Ly, et al.
(författare)
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Endpoint and Kinetic Approaches for Assessing Transfection Efficacy in Mammalian Cell Culture
- 2022. - 3
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Ingår i: Cell Penetrating Peptides. - New York : Humana Press. - 9781071617519 - 9781071617526 ; , s. 529-545
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Bokkapitel (refereegranskat)abstract
- The efficacy of transfection reagents and nanoparticles is often assessed by measuring levels of expressed reporter protein. Fluorescence and luminescence based assays provide sensitive, quantifiable and repeatable approaches. The genes expressing reporter protein can be integrated into the cells to create stable reporter cell lines or can be expressed from a transfected plasmid. Green fluorescent protein, luciferase, and secreted alkaline phosphatase are well-established reporters with versatile applications. Monitoring changes in live cells during and after transfection offer opportunities to reveal related mechanisms, efficacy, and bottlenecks of transfection. In this chapter, we describe the experimental setup and considerations for in vitro screening of delivery vectors. This can further be extended to measurements in reporter cell lines.
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