RNA interference opened new approaches for disease treatment but safe and efficient cell delivery remains a bottleneck. Extracellular vesicles (EVs) are known to naturally shuttle RNA. Due to their potent cell internalization and low‐cost scalability, milk‐derived EVs in particular are considered promising RNA delivery systems. However, low drug loading currently impedes their use. Here, innovative exogenous loading strategies for small interfering RNA (siRNA) are explored and systematically compared regarding encapsulation efficiency, loading capacity, and loading concentration. Firstly, siRNA is pre‐accumulated in liposomes or stabilized calcium phosphate nanoparticles (CaP‐NP). The selected systems, which exhibited neutral or negative zeta potentials, are then applied for EV loading. Secondly, EVs are concentrated and applied to protocols known for liposome loading: dehydration‐rehydration of vesicles, based on freeze‐drying, and mixing by dual asymmetric centrifugation (DAC) after ultracentrifugation. Additionally, DAC after EV ultracentrifugation is combined with CaP‐NP leading to a synergistic loading performance. The balance between energy input for siRNA loading and EV integrity is evaluated by monitoring the EV size, marker proteins, and morphology. For the EV‐based siRNA formulation via DAC plus CaP‐NP, EV properties are sufficiently maintained to protect the siRNA from degradation and deliver cell‐death siRNA dose‐dependently in Caco‐2 cells.

Convection-enhanced delivery (CED) has been introduced as a concept in cancer treatment to generate high local concentrations of anticancer therapeutics and overcome the limited diffusional distribution, e.g., in the brain. RNA interference provides interesting therapeutic options to fight cancer cells but requires nanoparticulate (NP) carriers with a size below 100 nm as well as a low zeta potential for CED application. In this study, we investigated calcium phosphate NPs (CaP-NPs) as siRNA carriers for CED application. Since CaP-NPs tend to aggregate, we introduced a new terpolymer (o14PEGMA(1:1:2.5) NH₃) for stabilization of CaP-NPs intended for delivery of siRNA to brain cancer cells. This small terpolymer provides PEG chains for steric stabilization, and a fat alcohol to improve interfacial activity, as well as maleic anhydrides that allow for both labeling and high affinity to Ca(II) in the hydrolyzed state. In a systematic approach, we varied the Ca/P ratio as well as the terpolymer concentration and successfully stabilized NPs with the desired properties. Labeling of the terpolymer with the fluorescent dye Cy5 revealed the terpolymer’s high affinity to CaP. Importantly, we also determined a high efficiency of siRNA binding to the NPs that caused very effective survivin siRNA silencing in F98 rat brain cancer cells. Cytotoxicity investigations with a standard cell line resulted in minor and transient effects; no adverse effects were observed in organotypic brain slice cultures. However, more specific cytotoxicity investigations are required. This study provides a systematic and mechanistic analysis characterizing the effects of the first oligomer of a new class of stabilizers for siRNA-loaded CaP-NPs.

The local release of complexed siRNA from biomaterials opens precisely targeted therapeutic options. In this study, complexed siRNA was loaded to gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA). We aggregated these siRNA-loaded cGM with human mesenchymal stem cells (hMSC) to microtissues and stimulated them with osteogenic supplements. An efficient knockdown of chordin, a BMP-2 antagonist, caused a remarkably increased alkaline phosphatase (ALP) activity in the microtissues. cGM, as a component of microtissues, mineralized in a differentiation medium within 8–9 days, both in the presence and in the absence of cells. In order to investigate the effects of our pre-differentiated and chordin-silenced microtissues on bone homeostasis, we simulated in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). We found enhanced ALP activity and osteoprotegerin (OPG) secretion in the model system compared to control microtissues. Our results suggest osteoanabolic effects of pre-differentiated and chordin-silenced microtissues.

Small interfering RNAs (siRNAs) have drawn particular attention for their ability to transiently and sequence‐specifically silence target genes, not only for systemic but also for localized application. For bone regeneration, targeting inhibitory regulators by siRNAs offers opportunities to improve osteogenic–angiogenic coupling. Conventional experimental models often oversimplify this interaction as they fail to capture these multicellular tissue dynamics. To address this, we established a human three‐dimensional co‐culture model composed of osteogenic and vascular microtissues embedded in fibrin hydrogels to investigate siRNA effects on microtissue interaction. Local siRNA delivery to microtissues was achieved by oligomer‐stabilized calcium phosphate nanoparticles (CaP‐NP) loaded onto cross‐linked gelatin microparticles (cGM). siRNA/CaP‐NP‐loaded cGM were assembled with human mesenchymal stem cells (hMSCs) to microtissues. This approach was demonstrated by silencing two antagonists with distinct expression profiles: Chordin, a low‐abundance BMP inhibitor, and WWP‐1, a highly expressed E3 ligase. Only Chordin siRNA improved the osteogenic‐vascular cross‐talk, whereas WWP‐1 siRNA effects were limited to osteogenic effects. Next‐generation sequencing (NGS) supported these results. We demonstrate that this co‐culture platform permits systematic investigation of siRNA‐mediated modulation of osteogenic–endothelial interactions, offering a relevant human model for preselecting therapeutic siRNA targets to advance vascularized bone tissue regeneration.