Targeted delivery and endosomal cellular uptake of DARPin-siRNA bioconjugates: Influence of linker stability on gene silencing

Specific cell targeting and efficient intracellular delivery are major hurdles for the widespread therapeutic use of nucleic acid technologies, particularly siRNA mediated gene silencing. To enable receptor-mediated cell-specific targeting, we designed a synthesis scheme that can be generically used to engineer Designed Ankyrin Repeat Protein (DARPin)-siRNA bioconjugates. Different linkers, including labile disulfide-, and more stable thiol-maleimide- and triazole- (click chemistry) tethers were employed. Crosslinkers were first attached to a 3’-terminal aminohexyl chain on the siRNA sense strands. On the protein side thiols of a C-terminal cysteine were used as anchoring site for disulfide- and thiol-maleimide conjugate formation, while strain-promoted azido-alkyne cycloadditions were carried out at a metabolically introduced N-terminal azidohomoalanine. After establishing efficient purification methods, highly pure products were obtained. Bioconjugates of EpCAM-targeted DARPins with siRNA directed at the luciferase gene were evaluated for cell-specific binding, uptake and gene silencing. As shown by flow cytometry and fluorescence microscopy, all constructs retained the highly specific and high-affinity antigen recognition properties of the native DARPin. As expected, internalization was observed only in EpCAM-positive cell lines, and predominantly endolysosomal localization was detected. Disulfide linked conjugates showed lower serum stability against cleavage at the linker and thus lower internalization into endosomes compared to thiol-maleimide- and triazole-linked conjugates, yet induced more pronounced gene silencing. This indicates that the siRNA payload needs to be liberated from the protein in the endosome. Our data confirm the promise of DARPin-siRNA bioconjugates for tumor targeting, but also identified endosomal retention and limited cytosolic escape of the siRNA as the rate-limiting step for more efficient gene silencing

Manufacturing of a Secretoneurin Drug Delivery System with Self-Assembled Protamine Nanoparticles by Titration

<div><p>Since therapeutic peptides and oligonucleotides are gathering interests as active pharmaceutical ingredients (APIs), nanoparticulate drug delivery systems are becoming of great importance. Thereby, the possibility to design drug delivery systems according to the therapeutic needs of APIs enhances clinical implementation. Over the last years, the focus of our group was laid on protamine-oligonucleotide-nanoparticles (so called proticles), however, the possibility to modify the size, zeta potential or loading efficiencies was limited. Therefore, at the present study we integrated a stepwise addition of protamine (titration) into the formation process of proticles loaded with the angiogenic neuropeptide secretoneurin (SN). A particle size around 130 nm was determined when proticles were assembled by the commonly used protamine addition at once. Through application of the protamine titration process it was possible to modify and adjust the particle size between approx. 120 and 1200 nm (dependent on mass ratio) without influencing the SN loading capacity. Dynamic light scattering pointed out that the difference in particle size was most probably the result of a secondary aggregation. Initially-formed particles of early stages in the titration process aggregated towards bigger assemblies. Atomic-force-microscopy images also revealed differences in morphology along with different particle size. In contrast, the SN loading was only influenced by the applied mass ratio, where a slight saturation effect was observable. Up to 65% of deployed SN could be imbedded into the proticle matrix. An in-vivo biodistribution study (i.m.) showed a retarded distribution of SN from the site of injection after the application of a SN-proticle formulation. Further, it was demonstrated that SN loaded proticles can be successfully freeze-dried and resuspended afterwards. To conclude, the integration of the protamine titration process offers new possibilities for the formulation of proticles in order to address key parameters of drug delivery systems as size, API loading or modified drug release.</p></div

Correlation of mean particle size and derived count rate throughout protamine titration process.

<p>Development of mean particle size (ZAve, blue lines) and derived count rate (red lines) for each mass ratio (captioned on the top left of each graph). Horizontal axis indicates the steps of protamine addition along the titration process. The results are given as mean values + standard deviation (dashed error bars refer to particle size).</p

AFM images of secretoneurin loaded proticles.

<p>Images recorded by atomic force microscopy showed spherical secretoneurin loaded proticles for mass ratio of 1 : 1 : 1.5 prepared by titration process (A) as well as by single protamine addition (B). Triangular clustered proticles were recorded for mass ratio 1 : 0.25 : 1.5 prepared by protamine addition at once (C).</p

Mean particle size of secretoneurin loaded proticles obtained by different mass ratios and preparation methods.

<p>The size of particles is given as mean hydrodynamic diameter (ZAve d.nm) separated by the applied preparation method (red bars: single protamine addition, blue bars: titration process) and mass ratio (1 = 100 μg/ml). (A) shows particles with 300 μg/ml protamine and (B) with 150 μg/ml. The results are given as mean values +/- standard deviation, each sample was measured at least in duplicate (number of samples are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164149#pone.0164149.t001" target="_blank">Table 1</a>). Statistical significance between preparation methods is marked as * (p<0.05), ** (p<0.01) and *** (p<0.001).</p

DLS measurements of SN loaded particles before and after lyophilization.

<p>DLS measurements of SN loaded particles before and after lyophilization.</p

Development of zeta potential throughout protamine titration.

<p>Particles were assembled without SN (blank marks) or 100 μg/ml SN (filled marks), as well as with 150 μg/ml protamine (blue lines) or 300 μg/ml (red lines). The ODN concentration was set to 100 μg/ml, each undiluted sample was measured at least in duplicate.</p