Dear Ryan S Pawell,

The following few publications may be helpful:

1- Research Article

Gene Therapy (2003) 10, 72–83. doi:10.1038/sj.gt.3301859

Enhanced cytosolic delivery of plasmid DNA by a sulfhydryl-activatable listeriolysin O/protamine conjugate utilizing cellular reducing potential

G Saito1, G L Amidon1 and K-D Lee1

1Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA

Correspondence: K-D Lee, Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church St., Ann Arbor, MI 48109-1065, USA

Received 1 March 2002; Accepted 16 June 2002.

Topof pageAbstract

Listeriolysin O (LLO), a sulfhydryl-activated pore-forming protein fromListeria monocytogenes, was tested and utilized for promoting plasmid DNA (pDNA) delivery into the cytosol of cells in culture. To render pDNA-complexing capability to LLO, the unique cysteine 484 of LLO was conjugated to polycationic peptide protamine (PN) at a 1:1 molar ratio through a reversible, endosome-labile disulfide bond. The sulfhydryl-oxidized LLO construct, LLO-s-s-PN, completely lacked its pore-forming activity, yet regained its original activity upon reduction. The enhanced cytosolic delivery using this construct therefore relies on the requisite reduction of the disulfide bond in LLO-s-s-PN by endogenous cellular reducing capacity. Condensed PN/pDNA complexes incorporating LLO-s-s-PN were tested for their enhanced gene delivery capability monitoring reporter gene expression in HEK293, RAW264.7, P388D1 cell lines and bone-marrow-derived macrophages in the presence of serum. Dramatic enhancement was observed for all tested complexes with varying weight ratios. The effect was most prominent at 0.64–0.80 (w/w) of PN/pDNA upon replacing 1–4% of PN with LLO-s-s-PN, resulting in approximately three orders of magnitude higher luciferase expression compared to PN/pDNA without apparent toxicity. These results demonstrate that incorporation of endosomolytic LLO into pDNA delivery systems in a controlled fashion is a promising approach of enhancing delivery into the cytosol of target cells in gene delivery strategies.

http://www.nature.com/gt/journal/v10/n1/full/3301859a.html

2-Angew Chem Int Ed Engl. 2015 Dec 7;54(50):15105-8. doi: 10.1002/anie.201505913. Epub 2015 Oct 30.

Detecting Cytosolic Peptide Delivery with the GFP Complementation Assay in the Low Micromolar Range.

Schmidt S1, Adjobo-Hermans MJ1, Wallbrecher R1, Verdurmen WP1,2, Bovée-Geurts PH1, van Oostrum J1, Milletti F3, Enderle T4, Brock R5.

Author information

Abstract

Transfection of cells with a plasmid encoding for the first ten strands of the GFP protein (GFP1-10) provides the means to detect cytosolic peptide import at low micromolar concentrations. Cytosolic import of the eleventh strand of the GFP protein either by electroporation or by cell-penetrating peptide-mediated import leads to formation of the full-length GFP protein and fluorescence. An increase in sensitivity is achieved through structural modifications of the peptide and the expression of GFP1-10 as a fusion protein with mCherry.

http://www.ncbi.nlm.nih.gov/pubmed/26515694

3- Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer

D Lechardeur1, K-J Sohn1, M Haardt1, P B Joshi2, M Monck2, R W Graham2, B Beatty3, J Squire3, H O'Brodovich4 and G L Lukacs1,3,a

Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting hurdles to gene transfer, utilizing a nonviral delivery system such as polycation-DNA complex. Following its internalization by endocytosis, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. However, the stability of plasmid DNA in the cytoplasm, that may play a determinant role in the transfection efficiency, is not known. The turnover of plasmid DNA, delivered by microinjection into the cytosol, was determined by fluorescence in situ hybridization (FISH) and quantitative single-cell fluorescence video-image analysis. Both single- and double-stranded circular plasmid DNA disappeared with an apparent half-life of 50-90 min from the cytoplasm of HeLa and COS cells, while the amount of co-injected dextran (MW 70000) remained unaltered. We propose that cytosolic nuclease(s) are responsible for the rapid degradation of plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of microinjected plasmid DNA was inhibited in cytosol-depleted cells or following the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent elimination of free 3'-OH ends could be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by selective permeabilization of the plasma membrane, exhibits divalent cation-dependent, thermolabile nuclease activity, determined by Southern blotting and 32P-release from end-labeled DNA. Collectively, these findings suggest that the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may constitute a previously unrecognized impediment for DNA translocation into the nucleus and a possible target to enhance the efficiency of gene delivery.

http://www.nature.com/gt/journal/v6/n4/full/3300867a.html

4-Detecting Cytosolic Peptide Delivery with the GFP Complementation Assay in the Low Micromolar Range

Transfection of cells with a plasmid encoding for the first ten strands of the GFP protein (GFP1-10) provides the means to detect cytosolic peptide import at low micromolar concentrations. Cytosolic import of the eleventh strand of the GFP protein either by electroporation or by cell-penetrating peptide-mediated import leads to formation of the full-length GFP protein and fluorescence. An increase in sensitivity is achieved through structural modifications of the peptide and the expression of GFP1-10 as a fusion protein with mCherry.

http://onlinelibrary.wiley.com/doi/10.1002/ange.201505913/full

5-Cytoplasmic delivery of proteins and DNA by pH-sensitive liposomes

The technique of introducing exogenous DNA into living cells, known as transfection, is a commonly used and very important approach in molecular biology. A number of methods have been developed to achieve this, 1-12 but none of them can likely be used in vivo studies. In this paper we would like to introduce a pH-sensitive immunoliposome delivery system that can efficiently deliver such macromolecules in vivo in a target-specific manner.

Mechanism of delivery

Immunoliposomes are lipid vesicles coated with antibodies that have been attached with hydrophobic anchors.13 These surface antibodies direct the specific interaction of liposomes with antigen-expressing cells. Previous studies revealed that the bound vesicles are taken up by cells through a receptor-mediated endocytotic process.14 To prevent liposomes and their contents from being delivered to the lysosomes for degradation, pH-sensitive immunoliposomes are developed to enhance a prelysosomal discharge of the liposome contents into the cellular cytoplasm.

It was found for the first time in this laboratory that liposomes composed of unsaturated phosphatidylethanolamine and a weakly acidic amphiphile, such as fatty acid, become destabilized and fusion-competent when the pH of the medium is reduced to below 6.5.15 If these types of liposomes are endocytosed into endosomes that have a pH range of 5 to 6.5,16,17 they will release their contents into cell cytoplasm by fusing with the endosome membrane from within and/or rupturing the endosome membrane.18 The prelysosomal discharge of the liposome contents significantly enhances the cytoplasmic delivery efficiency of the liposome for anti-tumor drugs,19toxin20 and DNA.18,21

http://www.fao.org/wairdocs/ilri/x5550e/x5550e0g.htm

Hoping this will be helpful,

Rafik