Fluorescent Peptide Dendrimers for siRNA Transfection: Tracking pH Responsive Aggregation, siRNA Binding, and Cell Penetration.

Transfecting nucleic acids into various cells is a key procedure in biological research also envisioned for therapeutic applications. In our effort to obtain simple reagents that would be readily accessible from commercial building blocks, we recently reported peptide dendrimers as single component siRNA transfection reagents accessible in pure form by solid-phase peptide synthesis. Here, we extend our studies of these dendrimers by identifying analogs bearing a coumarin or BODIPY fluorescent label in their core and displaying comparable siRNA transfection efficiencies, pH dependent aggregation, siRNA binding, and secondary structures. Fluorescence resonance energy transfer (FRET) studies show that the dendrimers are tightly associated with siRNA within the formed nanoparticles at pH 7.4 but are released into solution at pH 5.0 and can participate in endosome escape by destabilizing the membrane at this pH value. Colocalization studies furthermore suggest that peptide dendrimers and siRNA remain tightly associated throughout the transfection process.

Stereoselective pH Responsive Peptide Dendrimers for siRNA Transfection.

Transfecting nucleic acids into cells is an essential procedure in biological research usually performed using nonviral transfection reagents. Unfortunately, most transfection reagents have polymeric or undisclosed structures and require nonstandard synthetic procedures. Herein we report peptide dendrimers accessible as pure products from standard building blocks by solid-phase peptide synthesis and acting as nontoxic single component siRNA transfection reagents for a variety of cell lines with equal or better performance than the gold standard lipofectamine L2000. Structure-activity relationships and mechanistic studies illuminate their transfection mechanism in unprecedented detail. Stereoselective dendrimer aggregation via intermolecular ß-sheets at neutral pH enables siRNA complexation to form nanoparticles which enter cells by endocytosis. Endosome acidification triggers protonation of amino termini and rearrangement to an α-helical conformation forming smaller dendrimer/siRNA nanoparticles, which escape the endosome and release their siRNA cargo in the cytosol. Two particularly efficient d-enantiomeric dendrimers are proposed as new reference reagents for siRNA transfection.

Lipidated Peptide Dendrimers Killing Multidrug-Resistant Bacteria.

New antibiotics are urgently needed to address multidrug-resistant (MDR) bacteria. Herein we report that second-generation (G2) peptide dendrimers bearing a fatty acid chain at the dendrimer core efficiently kill Gram-negative bacteria including Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most problematic MDR bacteria worldwide. Our most active dendrimer TNS18 is also active against Gram-positive methicillin-resistant Staphylococcus aureus. Based on circular dichroism and molecular dynamics studies, we hypothesize that TNS18 adopts a hydrophobically collapsed conformation in water with the fatty acid chain backfolded onto the peptide dendrimer branches and that the dendrimer unfolds in contact with the membrane to expose its lipid chain and hydrophobic residues, thereby facilitating membrane disruption leading to rapid bacterial cell death. Dendrimer TNS18 shows promising in vivo activity against MDR clinical isolates of A. baumannii and Escherichia coli, suggesting that lipidated peptide dendrimers might become a new class of antibacterial agents.

Peptide Dendrimer-Lipid Conjugates as DNA and siRNA Transfection Reagents: Role of Charge Distribution Across Generations.

Transfection reagents are used to deliver DNA and siRNA into cells to achieve genetic manipulations, and may ultimately enable nonviral gene therapy. Progress in transfection reagents is limited by the fact that such reagents cannot be easily optimized due to their polymeric nature and/or difficult synthesis. We have developed a new class of well-defined and easily modifiable transfection reagents in the form of peptide dendrimers. These dendrimers self-assemble with DNA or siRNA and lipofectin to form nanoparticles which efficiently enter mammalian cells and liberate their nucleic acid cargo. By systematically modifying the amino acid sequence of our dendrimers we have found that their transfection efficiency depends on the distribution of positive charges and hydrophobic residues across the dendrimer branches. Positive charges present in all three generations lead to efficient DNA delivery, whereas siRNA delivery requires charges in the outer two generations combined with a hydrophobic dendrimer core.

Efficient Transfection of siRNA by Peptide Dendrimer-Lipid Conjugates.

Efficient delivery of small interfering RNA (siRNA) into cells is the basis of target-gene-specific silencing and, ultimately, gene therapy. However, current transfection reagents are relatively inefficient, and very few studies provide the sort of systematic understanding based on structure-activity relationships that would provide rationales for their improvement. This work established peptide dendrimers (administered with cationic lipids) as siRNA transfection reagents and recorded structure-activity relationships that highlighted the importance of positive charge distribution in the two outer layers and a hydrophobic core as key features for efficient performance. These dendrimer-based transfection reagents work as well as highly optimised commercial reagents, yet show less toxicity and fewer off-target effects. Additionally, the degrees of freedom in the synthetic procedure will allow the placing of decisive recognition features to enhance and fine-tune transfection and cell specificity in the future.

Enzymatic degradation pattern of polysorbate 20 impacts interfacial properties of monoclonal antibody formulations.

Polysorbate 20 (PS20) is widely used to maintain protein stability in biopharmaceutical formulations. However, PS20 is susceptible to hydrolytic degradation catalyzed by trace amounts of residual host cell proteins present in monoclonal antibody (mAb) formulations. The resulting loss of intact surfactant and the presence of PS20 degradation products, such as free fatty acids (FFAs), may impair protein stability. In this study, two hydrolytically-active immobilized lipases, which primarily targeted either monoester or higher-order ester species in PS20, were used to generate partially-degraded PS20. The impact of PS20 degradation pattern on critical micelle concentration (CMC), surface tension, interfacial rheology parameters and agitation protection was assessed. CMC was slightly increased upon monoester degradation, but significantly increased upon higher-order ester degradation. The PS20 degradation pattern also significantly impacted the dynamic surface tension of a mAb formulation, whereas changes in the equilibrium surface tension were mainly caused by the adsorption of FFAs onto the air-water interface. In an agitation protection study, monoester degradation resulted in the formation of soluble mAb aggregates and proteinaceous particles, suggesting that preferential degradation of PS20 monoester species can significantly impair mAb stability. Additional mAbs should be tested in the future to assess the impact of the protein format.

Lipophilic Peptide Dendrimers for Delivery of Splice-Switching Oligonucleotides.

Non-viral transfection reagents are continuously being developed in attempt to replace viral vectors. Among those non-viral vectors, dendrimers have gained increasing interest due to their unique molecular structure and multivalency. However, more improvements are still needed to achieve higher efficacy and lower toxicity. In this study, we have examined 18 peptide dendrimers conjugated to lipophilic moieties, such as fatty acids or hydrophobic amino acids, that were previously explored for siRNA. Reporter cells were employed to investigate the transfection of single strand splice-switching oligonucleotides (ONs) using these peptide dendrimers. Luciferase level changes reflecting efficiency varied with amino acid composition, stereochemistry, and complexation media used. 3rd generation peptide dendrimers with D-amino acid configuration were superior to L-form. Lead formulations with 3rd generation, D-amino acid peptide dendrimers increased the correction level of the delivered ON up to 93-fold over untreated HeLa Luc/705 cells with minimal toxicity. To stabilize the formed complexes, Polyvinyl alcohol 18 (PVA18) polymer was added. Although PVA18 addition increased activity, toxicity when using our best candidates G 2,3KL-(Leu)4 (D) and G 2,3KL-diPalmitamide (D) was observed. Our findings demonstrate the potential of lipid-conjugated, D-amino acid-containing peptide dendrimers to be utilized as an effective and safe delivery vector for splice-switching ONs.

Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor.

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials.

Novel peptide-dendrimer/lipid/oligonucleotide ternary complexes for efficient cellular uptake and improved splice-switching activity.

Despite the advances in gene therapy and in oligonucleotide (ON) chemistry, efficient cellular delivery remains an obstacle. Most current transfection reagents suffer from low efficacy or high cytotoxicity. In this report, we describe the synergism between lipid and dendrimer delivery vectors to enhance the transfection efficiency, while avoiding high toxicity. We screened a library of 20 peptide dendrimers representing three different generations and evaluated their capability to deliver a single-stranded splice-switching ON after formulating with lipids (DOTMA/DOPE). The transfection efficiency was analyzed in 5 reporter cell lines, in serum-free and serum conditions, and with 5 different formulation protocols. All formulations displayed low cytotoxicity to the majority of the tested cell lines. The complex sizes were < 200 nm; particle size distributions of effective mixtures were < 80 nm; and, the zeta potential was dependent on the formulation buffer used. The best dendrimer enhanced transfection in a HeLa reporter cell line by 30-fold compared to untreated cells under serum-free conditions. Interestingly, addition of sucrose to the formulation enabled - for the first time - peptide dendrimers/lipid complexes to efficiently deliver splice-switching ON in the presence of serum, reaching 40-fold increase in splice switching. Finally, in vivo studies highlighted the potential of these formulae to change the biodistribution pattern to be more towards the liver (90% of injected dose) compared to the kidneys (5% of injected dose) or to unformulated ON. This success encourages further development of peptide dendrimer complexes active in serum and future investigation of mechanisms behind the influence of additives on transfection efficacy.

Frontiers in Medicinal Chemistry 2017 in Bern, Switzerland.

Sharing capital ideas: The 2017 Frontiers in Medicinal Chemistry (FiMC) conference, organized jointly by the German Chemical Society, the German Pharmaceutical Society, and the Swiss Chemical Society, was held at the Department of Chemistry and Biochemistry of the University of Bern in February 2017. Herein we summarize the many conference highlights, and look forward to the next FiMC meeting, to be held in Jena (Germany) in March 2018.