Petra Kos

Solid-phase-assisted synthesis of targeting peptide–PEG–oligo(ethane amino)amides for receptor-mediated gene delivery

In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and α(v)β(3) integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer.

Sequence-defined four-arm oligo(ethanamino)amides for pDNA and siRNA delivery: Impact of building blocks on efficacy.

Cationic oligomers were assembled by solid-phase supported synthesis in few coupling steps based on C-terminal alanine and two lysine branchings, followed by elongation of the four arms with two to five repeats of artificial oligoamino acids containing the 1,2-diaminoethane motif, and ended by N-terminal cysteines or alanines. These sequence-defined oligomers, containing between 28 and 68 protonatable nitrogens, were evaluated for complex formation with plasmid DNA (pDNA) and short interfering RNA (siRNA), followed by reporter gene transfer and gene silencing experiments in Neuro2A cells. By two simple variations, the pDNA gene transfer activity could be thousand-fold improved, exceeding the gold standard linear PEI up to >50-fold. Firstly, the N-terminal cysteines introduced for bioreversible stabilization of polyplexes by internal disulfide links after complex formation greatly enhanced gene transfer. Secondly, variation of the artificial oligoamino acid building blocks containing either triethylene tetramine (Gtt), tetraethylene pentamine (Stp), or pentaethylene hexamine (Sph) disclosed a clear ranking in the order of Sph>Stp>>Gtt for both pDNA compaction and transfection activity. Extending the chain lengths of the arms beyond three building blocks had marginal impact on the performance. For the much smaller siRNA cargo, polyplex stabilization by cysteine disulfides presents a strict requirement. Sph and Stp based cysteine-ended four-arms displayed similar binding activity, with Stp providing best gene silencing efficiency.

Correlation of Length of Linear Oligo(ethanamino) Amides with Gene Transfer and Cytotoxicity

The optimization of synthetic carriers for gene transfer remains a major challenge. Cationic polymers such as polyethylenimine (PEI) often show increasing gene transfer activity with increasing molecular weight, but this favorable effect is accompanied by an undesired increase in cytotoxicity. Moreover, the polydispersity of polymers prevents accurate determination of optimum size. Herein we describe the step‐by‐step elongation of precise linear oligo(ethanamino) amides by making use of the artificial amino acid succinoyl‐tetraethylene pentamine (Stp) for solid‐phase‐assisted synthesis. This procedure enabled us to identify the optimal oligomer Stp30‐W (8.4 kDa) with a length of 30 Stp units, with which effective gene transfer occurs in the absence of cytotoxicity. The transfection efficiency of Stp30‐W exceeded that of standard linear PEI (22 kDa) by sixfold; nevertheless, Stp30‐W exhibited tenfold lower cytotoxicity. In addition to the lower molecular weight, the succinate spacer between the oligoamine units may also contribute to the favorable biocompatibility. The cytotoxicity of the cationic polymer PEI is a major concern for use as a carrier for gene delivery, so this comparison between linear PEI and the new Stp oligomers is particularly relevant.

Native chemical ligation for conversion of sequence-defined oligomers into targeted pDNA and siRNA carriers.

Native chemical ligation (NCL) was established for the conversion of sequence-defined oligomers of different topologies into targeted and PEG shielded pDNA and siRNA carriers. From an existing library of non-targeted oligoethanamino amides, six oligomers containing N-terminal cysteines were selected as cationic cores, to which monodisperse polyethylene glycol (PEG) containing terminal folic acid as targeting ligand (or terminal alanine as targeting negative control ligand) were attached by NCL. Ligated conjugates plus controls (in sum 18 oligomers) were evaluated for pDNA or siRNA gene delivery. Biophysical characteristics including nucleic acid binding in the absence or presence of serum, as well as biological activities in cellular uptake and gene transfer (or gene silencing, respectively) were determined. In most cases, the folic acid-PEG-ligated oligomers displayed a strongly improved cellular binding, uptake and gene transfer into receptor-positive KB cells as compared to the alanine-PEG controls. Changing the topological structures by increasing the number of cationic arms, adding tyrosine trimers as polyplex stabilizing domains, or histidines facilitating endosomal escape resulted in beneficial gene transfer characteristics. The screen revealed different requirements for pDNA and siRNA delivery. A folate-PEG ligated histidinylated four-arm oligomer was most effective for pDNA delivery but inactive for siRNA, whereas a folate-PEG-ligated three-arm oligomer with tyrosine trimer modifications was most effective in siRNA mediated gene silencing. The results demonstrate the site-selective NCL reaction as powerful method to modify existing oligomers. Thus multifunctional targeted carriers can be obtained with ease and used to identify lead structures for subsequent in vivo delivery.

Twin disulfides as opportunity for improving stability and transfection efficiency of oligoaminoethane polyplexes

The synthesis of precise gene delivery vehicles by solid-supported chemistry is an effective way to establish structure-activity relationships and optimize existing transfection carriers. Sequence-defined cationic oligomers with different topologies were modified with twin disulfide-forming cysteine-arginine-cysteine (CRC) motifs. The influence of this motif versus single disulfide on the biophysical properties and biological performance of polyplexes was investigated, with pDNA and siRNA as nucleic acid cargoes. Clear differences between structures with isolated cysteines and CRC motifs were observed with respect to properties like nucleic acid binding, serum stability, response to reducing agents, and gene transfer/silencing. The main observed effect of the CRC motif was to increase polyplex stability. The consequences for nucleic acid delivery were less predictable and depended on oligomer topology. For some oligomers intrinsically forming stable polyplexes (i.e., already in the absence of CRC motif), this further stabilization resulted in a reduction or even loss in transfection efficiency. For PEGylated and targeted oligomers with intrinsically less stable polyplex structures, this modification led to a significant enhancement in transfection efficiency.

Comb-like oligoaminoethane carriers: change in topology improves pDNA delivery.

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.

Dual-Targeted Polyplexes Based on Sequence-Defined Peptide-PEG-Oligoamino Amides

For active cell targeting, viruses frequently capitalize on dual-receptor binding. With the intention to mimic this natural process, a dual peptide-based approach for targeting cancer cells was evaluated. For this purpose, sequence-defined pDNA binding oligo (ethane amino) amides containing a PEG chain with a peptidic targeting ligand at its distal end were applied. Integrin receptor-directed cyclic peptide cRGDfk, transferrin receptor-addressing peptide B6, and epidermal growth factor receptor-targeting peptide GE11 were used in the study in DU145 prostate cancer cells that express all three receptors. Dual-receptor targeted pDNA polyplexes were designed by combining two of the above ligands at various ratios, in order to find an optimal ligand combination. Two polycation/pDNA ratios of nitrogen/phosphate (N/P) 6 and 12 were tested. Dual targeting effects were most pronounced at the lower N/P ratio and found for all three combinations. Cell binding studies and pDNA transfections revealed GE11 plus B6 as the most potent combination. In general, a good correlation of cell binding with gene transfer was observed. Interestingly, GE11 peptide-based polyplexes-mediated bimodal cell association profiles. In contrast, B6 ligand, cRGD ligand, and dual-targeted polyplexes triggered more homogenous monomodal cell binding characteristics.

Combinatorial Optimization of Sequence-Defined Oligo(ethanamino)amides for Folate Receptor-Targeted pDNA and siRNA Delivery.

Cationic polymers present a versatile platform for the nonviral delivery of therapeutic nucleic acids. In order to achieve effective nucleic acid transfer, polymeric carriers ought to comprise multiple functionalities. Precise chemistries for site-specific placements of the different delivery modules within the carriers present the basis for uncovering structure-activity relationships required for further optimization. Here we present the design and systematic evaluation of a library of 42 sequence-defined oligo(ethanamino)amides generated by solid-phase assisted syntheses. The carriers contained two- or four-arm topologies of different artificial oligoamino acid domains for nucleic acid complexation, terminated by cysteines for disulfide-triggered polyplex stabilization, linked with monodisperse polyethylene glycol (PEG) for surface shielding and terminal folic acid for receptor specific cellular uptake. Additional functional elements included histidines for endosomal escape and/or tyrosine trimers for enhanced hydrophobic polyplex stabilization. In vitro screening of the oligomer library identified a folate-PEG-linked two-arm oligocation structure comprising histidines and tyrosine trimers as the most effective class of carriers for the delivery of pDNA and siRNA.

Gene regulation by intracellular delivery and photodegradation of nanoparticles containing small interfering RNA

Although the use of small interfering RNA (siRNA) is a promising technique for gene regulation, spatiotemporal control of the effects of siRNA must be achieved if the technique is to be safe and practical. Here, a method for spatiotemporal regulation of genes with nanoparticles containing siRNA is reported. The siRNA is encapsulated in photodegradable nanoparticles that are internalized to SKOV3-Luc cells, where the siRNA is released from the nanoparticles by UV irradiation for 30 s. The encapsulated siRNA only shows no gene-silencing effects, but release of the siRNA upon UV radiation leads to sequence-specific silencing of the luciferase gene in the cells. These results indicate that photodegradable siRNA-containing nanoparticles can be useful for time- and space-dependent regulation of gene expression in cells.

Combining polyethylenimine and Fe(III) for mediating pDNA transfection

BACKGROUND The potential use of Fe(III) ions in biomedical applications may predict the interest of its combination with pDNA-PEI polyplexes. The present work aims at assessing the impact of this metal on pDNA complex properties. METHODS Variations in the formation of complexes were imposed by using two types of biological buffers at different salt conditions. The incorporation of pDNA in complexes was characterised by gel electrophoresis and dynamic light scattering. Transfection efficiency and cytotoxicity were evaluated in HeLa and HUH-7 cell lines, supported by flow cytometry assays. RESULTS Fe(III) enhances pDNA incorporation in the complex, irrespective of the buffer used. Transfection studies reveal that the addition of Fe(III) to complexes at low ionic strength reduces gene transfection, while those prepared under high salt content do not affect or, in a specific case, increase gene transfection up to 5 times. This increase may be a consequence of a favoured interaction of polyplexes with cell membrane and uptake. At low salt conditions, results attained with chloroquine indicate that the metal may inhibit polyplex endosomal escape. A reduction on the amount of PEI (N/P 5) formed at intermediary ionic strength, complemented by Fe(III), reduces the size of complexes while maintaining a transfection efficiency similar to that obtained to N/P 6. CONCLUSIONS Fe(III) emerges as a good supporting condensing agent to modulate pDNA-PEI properties, including condensation, size and cytotoxicity, without a large penalty on gene transfection. GENERAL SIGNIFICANCE This study highlights important aspects that govern pDNA transfection and elucidates the benefits of incorporating the versatile Fe(III) in a gene delivery system.