6 research outputs found
Enhancing Endosomal Escape of Transduced Proteins by Photochemical Internalisation
<div><p>Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of <em>in vitro</em> and <em>in vivo</em> assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments <em>in vitro.</em></p> </div
Fluorescence signal per cell after transduction.
<p>Sarcoma cells were transduced with Atto488-BSA using different transduction reagents. The fluorescence was measured in a flow cytometer, untreated cells were used as control and standard endocytosis was measured by treating the cells with 2 µg Atto488-BSA containing DMEM. Error bars represent the 95% confidence intervals of the means depicted. Measurements were set to 20000 gated events.</p
Change of signal distribution after PCI treatment.
<p>The median of the pixel gray values was calculated in fluorescence images of Atto488-BSA transduced sarcoma cells using different transduction reagents. Single cells were defined as region of interest in the ImageJ software and the median of the gray values was compared between cells before and after PCI treatment as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052473#s2" target="_blank">Methods</a>. The means of the medians of the gray values before and after PCI treatment are given. Error bars represent the 95% confidence intervals (n = 10). CPP-based transduction reagents (Proteoducin and Chariot) show high signal distribution changes. Reagents based on lipids (Proteofectene, Lipodin-Pro, ProJect and Pro-DeliverIN) also show changes but with partially lower significance. Reagents described as non-lipid or cationic polymers (TransPassP and TurboFect) did not lead to a signal spreading as a reason of a PCI treatment. The cytosolic signals produced by transduction with the endosomolytic reagent Endo-Porter was increased significantly by further disruption of endo- and lysosomes by PCI treatment.</p
Fluorescence signal per lysate after transduction.
<p>The fluorescence intensity of the lysed cells per well was measured in a multiwell fluorescence reader. The intensities were normalized to the mean intensity produced by the transfection using Proteoducin as transduction reagent. The sarcoma cells were transduced with Atto488-BSA using different transduction reagents. Untreated sarcoma cells were measured as controls. As a control for standard endosomal uptake, sarcoma cells were treated with DMEM containing 0.5 µg Atto488-BSA without a transduction reagent (termed as Atto488-BSA). Error bars represent the 95% confidence intervals of the means. Each transduction reagent was tested in at least 6 independent experiments.</p
Microwave-Assisted Desulfonylation of Polysulfonamides toward Polypropylenimine
Linear
polyethylenimine (L-PEI) has been the gold standard for
gene delivery and is typically prepared by hydrolysis from polyÂ(2-oxazoline)Âs.
Recently, also the anionic polymerization of activated aziridines
was reported as a potential pathway toward linear and well-defined
polyamines. However, only sulfonamide-activated aziridines so far
undergo the living anionic polymerization and their desulfonylation
was only reported scarcely. This is mainly due to the relatively high
stability of the sulfonamides and the drastic change in solubility
during the desulfonylation. Herein, we investigated the desulfonylation
of such polyÂ(aziridine)Âs prepared from tosylated or mesylated propyleneimine
to afford linear polypropylenimine (L-PPI) as an alternative to L-PEI.
Different desulfonylation strategies for tosylated (Ts) and mesylated
(Ms) PPI were studied. The reductive cleavage of the sulfonamide with
sodium bisÂ(2-methoxy ethoxy) aluminum hydride yielded 80% of deprotected
amine groups. Quantitative conversion to L-PPI was obtained, when
the tosylated PPI was hydrolyzed under acidic conditions with <i>p</i>TsOH under microwave (MW) irradiation. The same treatment
removed 90% of the mesyl groups from the mesylated PPI analog. The
MW-assisted acidic hydrolysis represents a fast, inexpensive and easy
approach in comparison to other methods, where complex reaction conditions
and tedious purifications are major drawbacks, however some chain
scission may occur. The high purity of the obtained products, in combination
with the versatility of the activated aziridine chemistry, demonstrate
many advantages of our strategy, especially for future biomedical
implementations
Chemoselective Dual Labeling of Native and Recombinant Proteins
The
attachment of two different functionalities in a site-selective
fashion represents a great challenge in protein chemistry. We report
site specific dual functionalizations of peptides and proteins capitalizing
on reactivity differences of cysteines in their free (thiol) and protected,
oxidized (disulfide) forms. The dual functionalization of interleukin
2 and EYFP proceeded with no loss of bioactivity in a stepwise fashion
applying maleimide and disulfide rebridging allyl-sulfone groups.
In order to ensure broader applicability of the functionalization
strategy, a novel, short peptide sequence that introduces a disulfide
bridge was designed and site-selective dual labeling in the presence
of biogenic groups was successfully demonstrated