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When optimizing nanocarriers, structural motifs that are beneficial for the respective type of cargo need to be identified. Here, succinoyl tetraethylene pentamine (Stp)-based lipo-oligoaminoamides (OAAs) were optimized for the delivery of plasmid DNA (pDNA). Structural variations comprised saturated fatty acids with chain lengths between C2 and C18 and terminal cysteines as units promoting nanoparticle stabilization, histidines for endosomal buffering, and disulfide building blocks for redox-sensitive release. Biophysical and tumor cell culture screening established clear-cut relationships between lipo-OAAs and characteristics of the formed pDNA complexes. Based on the optimized alternating Stp-histidine backbones, lipo-OAAs containing fatty acids with chain lengths around C6 to C10 displayed maximum gene transfer with around 500-fold higher gene expression than that of C18 lipo-OAA analogues. Promising lipo-OAAs, however, showed only moderate in vivo efficiency. In vitro testing in 90% full serum, revealing considerable inhibition of lytic and gene-transfer activity, was found as a new screening model predictive for intravenous applications in vivo.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) system offers great opportunities for the treatment of numerous diseases by precise modification of the genome. The functional unit of the system is represented by Cas9/sgRNA ribonucleoproteins (RNP), which mediate sequence‐specific cleavage of DNA. For therapeutic applications, efficient and cell‐specific transport into target cells is essential. Here, Cas9 RNP nanocarriers are described, which are based on lipid‐modified oligoamino amides and folic acid (FolA)‐PEG to realize receptor‐mediated uptake and gene editing in cancer cells. In vitro studies confirm strongly enhanced potency of receptor‐mediated delivery, and the nanocarriers enable efficient knockout of GFP and two immune checkpoint genes, PD‐L1 and PVR, at low nanomolar concentrations. Compared with non‐targeted nanoparticles, FolA‐modified nanocarriers achieve substantially higher gene editing including dual PD‐L1/PVR gene disruption after injection into CT26 tumors in vivo. In the syngeneic mouse model, dual disruption of PD‐L1 and PVR leads to CD8+ T cell recruitment and distinct CT26 tumor growth inhibition, clearly superior to the individual knockouts alone. The reported Cas9 RNP nanocarriers represent a versatile platform for potent and receptor‐specific gene editing. In addition, the study demonstrates a promising strategy for cancer immunotherapy by permanent and combined immune checkpoint disruption.

Acquired drug resistance constitutes a serious obstacle to the successful therapy of cancer. In the process of therapy resistance, microRNAs can play important roles. In order to combat resistance formation and to improve the efficacy of chemotherapeutics, the mechanisms of the multifaceted hsa-miR−200c on drug resistance were elucidated. Upon knockout of hsa-miR−200c in breast carcinoma cells, a proteomic approach identified altered expression of glutathione S-transferases (GSTs) when cells were treated with the chemotherapeutic drug doxorubicin. In different hsa-miR−200c expression systems, such as knockout, inducible sponge and inducible overexpression, the differential expression of all members of the GST family was evaluated. Expression of hsa-miR−200c in cancer cells led to the repression of a multitude of these GSTs and as consequence, enhanced drug-induced tumor cell death which was evaluated for two chemotherapeutic drugs. Additionally, the influence of hsa-miR−200c on the glutathione pathway, which is part of the phase II detoxification mechanism, was investigated. Finally, the long-term effects of hsa-miR−200c on drug efficacy were studied in vitro and in vivo. Upon doxycycline induction of hsa-miR−200c, MDA-MB 231 xenograft mouse models revealed a strongly reduced tumor growth and an enhanced treatment response to doxorubicin. A combined treatment of these tumors with hsa-miR−200c and doxorubicin resulted in complete regression of the tumor in 60% of the animals. These results identify hsa-miR−200c as an important player regulating the cellular phase II detoxification, thus sensitizing cancer cells not expressing this microRNA to chemotherapeutics and reversing drug resistance through suppression of GSTs.

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