47 research outputs found

    Acid-Labile Traceless Click Linker for Protein Transduction

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    Intracellular delivery of active proteins presents an interesting approach in research and therapy. We created a protein transduction shuttle based on a new traceless click linker that combines the advantages of click reactions with implementation of reversible pH-sensitive bonds. The azidomethyl-methylmaleic anhydride (AzMMMan) linker was found compatible with different click chemistries, demonstrated in bioreversible protein modification with dyes, polyethylene glycol, or a transduction carrier. Linkages were stable at physiological pH but reversible at the mild acidic pH of endosomes or lysosomes. We show that pH-reversible attachment of a defined endosome-destabilizing three-arm oligo­(ethane amino)­amide carrier generates an effective shuttle for protein delivery. The cargo protein nlsEGFP, when coupled via the traceless AzMMMan linker, experiences efficient cellular uptake and endosomal escape into the cytosol, followed by import into the nucleus. In contrast, irreversible linkage to the same shuttle hampers nuclear delivery of nlsEGFP which after uptake remains trapped in the cytosol. Successful intracellular delivery of bioactive ß-galactosidase as a model enzyme was also demonstrated using the pH-controlled shuttle system

    Sequence Defined Disulfide-Linked Shuttle for Strongly Enhanced Intracellular Protein Delivery

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    Intracellular protein transduction technology is opening the door for a promising alternative to gene therapy. Techniques have to address all critical steps, like efficient cell uptake, endolysosomal escape, low toxicity, while maintaining full functional activity of the delivered protein. Here, we present the use of a chemically precise, structure defined three-arm cationic oligomer carrier molecule for protein delivery. This carrier of exact and low molecular weight combines good cellular uptake with efficient endosomal escape and low toxicity. The protein cargo is covalently attached by a bioreversible disulfide linkage. Murine 3T3 fibroblasts could be transduced very efficiently with cargo nlsEGFP, which was tagged with a nuclear localization signal. We could show subcellular delivery of the nlsEGFP to the nucleus, confirming cytosolic delivery and expected subsequent subcellular trafficking. Transfection efficiency was concentration-dependent in a directly linear mode and 20-fold higher in comparison with HIV-TAT-nlsEGFP containing a functional TAT transduction domain. Furthermore, β-galactosidase as a model enzyme cargo, modified with the carrier oligomer, was transduced into neuroblastoma cells in enzymatically active form

    New Sequence-Defined Polyaminoamides with Tailored Endosomolytic Properties for Plasmid DNA Delivery

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    Heterogeneity of polymeric carriers is one of the most elusive obstacles in the development of nonviral gene delivery systems, concealing interaction mechanisms and limiting the use of structure–activity relationship studies. In this report, novel sequence-defined polyaminoamides, prepared by solid-phase assisted synthesis, were used to establish first structure–activity relationships for polymer-based plasmid DNA delivery. By combining a cationic building block with hydrophobic modifications and bioreversible disulfide cross-linking sites, transfection polymers with tailored lytic and DNA binding properties were designed. These polymers demonstrated clear correlation between structure and performance in lysis and DNA binding assays. In vitro studies showed negligible toxicity and highly efficient gene transfer, demonstrating the potential of this platform in the fast, combinatorial development of new transfection polymers

    Comb-Like Oligoaminoethane Carriers: Change in Topology Improves pDNA Delivery

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    Establishing precise structure–activity relationships is important for the optimization of synthetic carriers for gene delivery. Sequence-defined oligomers with branched or linear shapes were synthesized to investigate the influence of topology on their biophysical properties and biological performance. Comb-like structures were synthesized consisting of an oligolysine peptide backbone modified at the ε-amino groups with four different artificial oligoamino acids, succinyl-diethylene triamine (Sdt), succinyl-triethylene tetramine (Stt), succinyl-tetraethylene pentamine (Stp), and succinyl-pentaethylene hexamine (Sph). Optionally the amino acids histidine and alanine were inserted into the oligolysine backbone to assess a possible buffer or spacer effect. After the evaluation of biophysical properties, the best performing oligomers, containing the Stp or Sph building blocks, were compared to corresponding linear oligomers where Stp or Sph are directly integrated into the linear oligolysine row. Clear differences between the comb and linear carriers were observed in the comparison of properties such as DNA complexation ability, buffer capacity, cellular association and internalization, and gene transfer. For the Stp containing structures, the comb topology mediated an increased buffer capacity at endosomal pH. For the Sph containing structures, in sharp contrast, the linear topology displayed advantageous endosomal buffering. Interestingly, for both Stp and Sph carriers, the comb in comparison to the linear topologies mediated a higher overall cellular uptake despite a lower cell association. For Stp combs, the combined advantage in both buffering and cellular uptake resulted in a strong (10- to >100-fold) increase in DNA transfection efficiency. In the case of Sph carriers, comb topology mediated only moderately (maximum 4-fold) enhanced gene transfer over the linear topology

    (poly IC)PEI-PEG-EGF(+PEI-Mel) Complexes Selectively Kill U87MGwtEGFR Cells

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    <div><p>(A) Cells were seeded in duplicate onto a 96-well plate at a density of 5,000 cells in 0.2 ml of medium per well and grown overnight. Cells were then transfected as described [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030006#pmed-0030006-b006" target="_blank">6</a>,<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030006#pmed-0030006-b009" target="_blank">9</a>] with poly IC at the indicated concentrations using either PEI-PEG-EGF or PEI-PEG-EGF+PEI-Mel (w/w ratio PEI-PEG-EGF:PEI-Mel = 1:10) complexes. Viability was measured by the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions, at 1 h after transfection.</p> <p>(B and C) Fast induction of apoptosis by (poly IC)PEI-PEG-EGF+PEI-Mel complexes. Apoptotic death was detected 1 h after transfection by Annexin (B) and TUNEL (C) assays as described in Methods.</p></div

    Cell Killing Mechanisms

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    <div><p>(A) Protection of the cells by 2-AP. Cells were grown as in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030006#pmed-0030006-g001" target="_blank">Figure 1</a>A. Cells were then transfected with poly IC at the indicated concentrations using PEI-PEG-EGF+PEI-Mel complexes. Where indicated, 2-AP (5 mM) was added 18 h before transfection and the medium was replaced every 24 h with medium containing fresh 2-AP. Viability was measured by the Methylene Blue assay [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030006#pmed-0030006-b008" target="_blank">8</a>].</p> <p>(B) In vitro bystander effect. U87MGwtEGFR cells were grown and transfected as described in Methods. U87MG and U87MGΔEGFR “indicator” cells were grown in duplicates in 96-well plates. Medium of the “indicator cells” was then partially replaced by the medium collected from the transfected (<b>+poly IC</b>) or untransfected (<b>-poly IC</b>) U87MGwtEGFR cells (<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030006#s2" target="_blank">Methods</a>). Where indicated the medium was preincubated with neutralizing polyclonal anti IFNα antibody. In <b>NT</b> samples, medium was not replaced.</p> <p>(C) A total of 4,000 U87MGwtEGFR and U87MGΔEGFR cells were seeded in duplicate onto a 96-well plate at the indicated ratios and grown overnight. Cells were then transfected with poly IC at the indicated concentrations using PEI-PEG-EGF+PEI-Mel conjugates. Cell survival was measured by the Methylene Blue assay 96 h after transfection.</p></div

    Distribution of the Complexes

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    <p>Cells were seeded into 6-cm plates at a density of 300,000 cells in 2 ml of medium per plate and grown overnight. Cells were then transfected with the fluorescently labeled poly IC (5 ÎĽg/ml) using the indicated conjugates. After 4 h, cells were analyzed by fluorescent microscopy (<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030006#s2" target="_blank">Methods</a>) for selectivity of the transfection (A) and intracellular distribution of the complex in the U87MGwtEGFR cells (B).</p

    Targeted Poly IC Eliminates Three Types of EGFR Over-Expressing Tumors

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    <div><p>(A) (poly IC)Mel-PEI-PEG-EGF (MPPE) complexes prolong survival of mice bearing large intracranial U87MGwtEGFR xenografts. Cells were implanted into the brains of 16 mice. 15 d later, two animals were sacrificed to measure the tumors (<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030006#s2" target="_blank">Methods</a>). Other animals received the indicated treatments. The daily doses of complexes were similar to the doses in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030006#pmed-0030006-g003" target="_blank">Figure 3</a>A and <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030006#pmed-0030006-g003" target="_blank">3</a>B. Survival of the animals was analyzed as above.</p> <p>(B) Formulated poly IC selectively kills A431 and MDA-MB-468 cells. Cells were seeded in duplicate onto a 96-well plate at a density of 5,000 cells in 0.2 ml of medium per well and grown overnight. Cells were then transfected as described [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030006#pmed-0030006-b006" target="_blank">6</a>,<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030006#pmed-0030006-b009" target="_blank">9</a>] with poly IC at the indicated concentrations using either MPPE or PEI-PEG-EGF+PEI-Mel. Cell survival was measured by the Methylene blue assay at 48 h after transfection.</p> <p>(C) (poly IC)MPPE complexes eliminate A431 and MDA-MB-468 xenografts in mice. A431 and MDA-MB-468 tumors were established and treated with formulated poly IC as described in Methods. Tumors were measured daily. Control animals were euthanized at day 33 after treatment initiation. Poly IC treated mice were kept alive to detect possible late recurrence of the tumors.</p></div

    TrkB and Bmi1 protein expression is hampered by overexpression of miR-200c in MDA-MB 436 cells.

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    <p>Total cell lysates of pre-miR-200c (pre) and scrambled control (scr) transfected MDA-MB 436 cells were subjected to western blot analysis and quantitative RT-PCR to determine expression of A) TrkB (gp145 and gp95) protein, B) TrkB mRNA C) p-Akt protein either after treatment with 0.5 µM doxorubicin for 24 hours (right panel) or untreated (left panel), D) Bmi1 protein, E) Bmi1 mRNA and F) p53 protein. α-Tubulin or Actin was used as loading control. Western blot quantification of three independent experiments was carried out by analyzing the relative intensities (rel. int.) of TrkB or Bmi1 normalized to the rel. int. of α-Tubulin or Actin using ImageJ software. For quantitative RT-PCR TrkB and Bmi1 expressions were normalized to GAPDH as reference and presented as ratio. A student’s t-test was performed to assess statistical significance. (ns = not significant; *p<0.05) DXR = doxorubicin.</p

    Molecular evolution of breast cancer cells leads to a chemoresistant phenotype and down-regulation of miR-200c.

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    <p>A) Molecular Evolution Assay. The epithelial breast cancer cell line BT474 was sequentially treated with chemotherapy. Cells were treated with 50 nM doxorubicin for 72 hours. Subsequently, medium was replaced by fresh medium until cells recovered and reached a confluency of 80%. Finally, cells were splitted for RNA isolation, cell lysis (protein), cytotoxicity assays and the next treatment round. R0 represents the untreated control cell line, whereas R1, R2, R3 and R4 represents BT474 cells that are treated for one, two, three and four times, respectively. B) Susceptibility to doxorubicin treatment. BT474 cells of R1 to R4 were treated with 0.1 and 10 µM doxorubicin for 72 hours. A CellTiter Glo assay was carried out to determine cell viability. Results are indicated as percentage of viable cells normalized to mock treated cells. C) Cell morphology of untreated and treated BT474 cells. Microscopic pictures (phase contrast) were taken from untreated BT474 cells (R0) and from doxorubicin treated and recovered cells of R4. D) Epithelial and mesenchymal marker expression in BT474 cells of R0, R2 and R4 of the Molecular Evolution Assay. E-Cadherin and Vimentin protein levels were determined by western blot analysis. Actin was used as loading control. E) miR-200c expression in BT474 cells that have undergone molecular evolution. Quantitative RT-PCR was performed to analyze miR-200c levels in BT474 cells of R1 to R4. miR-200c expression was thereby normalized to miR-191. Results are depicted as fold expression compared to the untreated control cell line (R0). Experiments were done in triplicates. For statistical analysis a student’s t-test was performed. (*p<0.05; **p<0.01; ***p<0.001).</p
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