Abstract
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.
Highlights
The formulation of an effective nanoparticulate drug delivery system requires consideration of various physicochemical properties
The characterization of the SN-proticle formulations focused on the mean particle size, the particle size distribution and SN loading
Various mass ratios were applied by adding protamine in a single step or by a stepwise titration process in order to verify the effect of the manufacturing process and the mass ratio on size and loading efficiencies
Summary
The formulation of an effective nanoparticulate drug delivery system requires consideration of various physicochemical properties. The therapeutic effect of an active pharmaceutical ingredient (API) is strongly influenced by the size and the surface charge of the delivery system [1, 2] as well as from the hydrophobicity [3] and morphology [4] These properties are controlling the bioavailability of the API [5] as well as the release profile from the delivery system [3], and will further affect cellular uptake [1, 2] and targeting issues. The possibility to provide various proticle formulations differing in size and loading capacity was restricted to a formation method by self-assembly To overcome these limitation, at our present study we describe a stepwise addition of protamine (titration) for the formulation of secretoneurin (SN) loaded proticles. A CpG-control ODN was used at all proticle formulations as negatively charged component for particle assembly, because our earlier studies already revealed that proticles consisting of CpG-control ODNs and protamine do not show immunostimulatory side effects at all [7]
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