**4. Conclusions**

In this study, we exploited the tunability of MSNs to synthesize a series of core-shell MSNs with different particle sizes between 60 to 160 nm for studying the size effect of these carriers on antitumoral miRNA delivery. All other properties of the delivery vehicles, including surface area, pore size and zeta

potential were kept comparable. The nanoparticles were capped with a positively charged block copolymer 454 equipped with the targeting agen<sup>t</sup> Mal-PEG-GE11. Since gene silencing was only observed after capping the nanocarriers with this 454 polymer, we conclude that it is essential for the endosomal escape by destabilizing the endosomal membrane. It was shown that the targeting ligand GE11 enhances a receptor-mediated uptake. After capping, the MSN-454-GE11 vehicles were used for a systematic investigation of size-dependent gene silencing. While smaller particles did not lead to significant effects, MSN-454-GE11 with a size of 160 nm showed a remarkable gene knockdown efficacy and antitumoral effects such as a decreased migration and changes in cell cycle. Overall, we observed the fastest cellular internalization as well as the best knock-down efficacies with MSN-454-GE11 sized 160 nm. In contrast to FACS results that indicated a particle association with the cells independent of MSN particle size, we found with statistically evaluated image analysis that only the largest particles are truly internalized in the cells after short incubation times. Thus, cell studies as performed here, aiming to simulate dynamic conditions of in vivo drug delivery by washing the incubated cells after a short incubation time, might discriminate against all particles that are not well attached to the cell surface. Fast internalized particles are thus the winner. We hypothesize that due to their size, they expose a larger contact area to the cell membrane and simultaneously a larger number of targeting ligands, which, when spaced just right, allow for a maximum degree of endocytosis. Our study shows that fast cellular internalization is essential for a successful downregulation. In summary, the nanoscale MSN160 nm-454-GE11 vehicles show the most promising potential for future in vivo biomedical applications.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/12/6/505/s1, Figure S1: DLS measurements of MSN samples, Figure S2: TGA, Figure S3: Structure of copolymer 454, Figure S4: Zeta potential titration curves of pure MSN samples, Figure S5: 454-GE11 calibration curve, Figure S6: DLS measurements of MSN-454-GE11 samples, Figure S7: Quantification of FACS data shown in Figure 4b, Figure S8: Confocal fluorescence microscopy images, Figure S9: Quantification of the cellular uptake of MSN-454-GE11, Figure S10: Gene-silencing assay, Figure S11: MTT cell viability study, Figure S12: Inhibition results, Figure S13: FACS of internalization of untargeted nanoparticles, Table S1: Estimated capping amount of 454-GE11 for different MSN samples.

**Author Contributions:** Conceptualization, T.B., E.W., K.M.; Measurements and Data Analysis, L.H., W.Z., S.R., H.E.; Writing—Original Draft Preparation, L.H.; Writing—Review & Editing, K.M., T.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** Financial support from the DFG (SFB 1032), the Excellence Cluster Nanosystems Initiative Munich (NIM), the Center for NanoScience Munich (CeNS), as well as Ludwig-Maximilians-Universität (LMUexcellent funds) is gratefully acknowledged.

**Acknowledgments:** We thank Steffen Schmidt for electron microscopy.

**Conflicts of Interest:** Authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
