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Extracellular vesicles (ECVs) are secreted cell-derived membrane particles involved in intercellular signaling and cell-cell communication. By transporting various bio-macromolecules, ECVs and in particular exosomes are relevant in various (patho-) physiological processes. ECVs are also released by cancer cells and can confer pro-tumorigenic effects. Their target cell tropism, effects on proliferation rates, natural stability in blood and immunotolerance makes ECVs particularly interesting as delivery vehicles. Polyethylenimines (PEIs) are linear or branched polymers which are capable of forming non-covalent complexes with small RNA molecules including siRNAs or antimiRs, for their delivery in vitro and in vivo. This study explores for the first time the combination of PEI-based nanoparticles with naturally occurring ECVs from different cell lines, for the delivery of small RNAs. ECV-modified PEI/siRNA complexes are analyzed by electron microscopy vs. ECV or complex alone. On the functional side, we demonstrate increased knockdown efficacy and storage stability of PEI/siRNA complexes upon their modification with ECVs. This is paralleled by enhanced tumor cell-inhibition by ECV-modified PEI/siRNA complexes targeting Survivin. Pre-treatment with various inhibitors of cellular internalization reveals alterations in cellular uptake mechanisms and biological activities of PEI/siRNA complexes upon their ECV modification. Extending our studies towards PEI-complexed antimiRs against miR-155 or miR-1246, dose-dependent cellular and molecular effects are enhanced in ECV-modified complexes, based on the de-repression of direct miRNA target genes. Differences between ECVs from different cell lines are observed regarding their capacity of enhancing PEI/siRNA efficacies, independent of the target cell line for transfection. Finally, an in vivo therapy study in mice bearing s.c. PC3 prostate carcinoma xenografts reveals marked inhibition of tumor growth upon treatment with ECV PC3 -modified PEI/siSurvivin complexes, based on profound target gene knockdown. We conclude that ECV-modification enhances the activity of PEI-based complexes, by altering pivotal physicochemical and biological nanoparticle properties., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Therapeutic applications of RNA interference (RNAi) require efficient siRNA delivery strategies in vivo. Combining lipid-based carriers with polymeric nanoparticles offers the favorable properties of both systems. This is the first study to explore polyethylenimine-based lipopolyplexes comprising a low-molecular weight PEI and the phospholipid DPPC for therapeutic siRNA use. Lipopolyplex structures are analyzed by electron microscopy. Biological efficacies are demonstrated in vitro by cellular uptake, knockdown of the target oncogene survivin, and concomitant cell growth inhibition. Upon systemic administration in tumor-bearing mice, here performed by intraperitoneal (i.p.) injection, radioactive biodistribution assays show lipopolyplex-mediated delivery of intact siRNAs. Absence of blood serum parameter alterations, erythrocyte aggregation or immunostimulation, and the observation of animal well-being and stable body weight confirm biocompatibility. Exploring therapeutic efficacies in a preclinical model, a considerable inhibition of prostate carcinoma xenograft growth is achieved, paralleled by an ~65% survivin knockdown in the tumors. We, thus, demonstrate that PEI-based lipopolyplexes represent an efficient platform for therapeutic use of small RNAs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The delivery of nucleic acids, particularly of small RNA molecules like siRNAs for the induction of RNA interference (RNAi), still represents a major hurdle with regard to their application in vivo. Possible therapeutic applications thus rely on the development of efficient non-viral gene delivery vectors. While low molecular weight polyethylenimines (PEIs) have been successfully explored, the introduction of chemical modifications offers an avenue towards the development of more efficient vectors. In this paper, we describe the synthesis of a novel tyrosine-modified low-molecular weight polyethylenimine (P10Y) for efficient siRNA complexation and delivery. The comparison with the respective parent PEI reveals that knockdown efficacies are considerably enhanced by the tyrosine modification, as determined in different reporter cell lines, without appreciable cytotoxicity. We furthermore identify optimal conditions for complex preparation as well as for storing or lyophilization of the complexes without loss of biological activity. Beyond reporter cell lines, P10Y/siRNA complexes mediate the efficient knockdown of endogenous target genes and, upon knockdown of the anti-apoptotic oncogene survivin, tumor cell inhibitory effects in different carcinoma cell lines. Pushing the system further towards its therapeutic in vivo application, we demonstrate in mice the delivery of intact siRNAs and distinct biodistribution profiles upon systemic (intravenous or intraperitoneal) injection. No adverse effects (hepatotoxicity, immunostimulation/alterations in immunophenotype, weight loss) are observed. More importantly, profound tumor-inhibitory effects in a melanoma xenograft mouse model are observed upon systemic application of P10Y/siRNA complexes for survivin knockdown, indicating the therapeutic efficacy of P10Y/siRNA complexes. Taken together, we (i) establish tyrosine-modified PEI (P10Y) as efficient platform for siRNA delivery in vitro and in vivo, (ii) identify optimal preparation and storage conditions as well as (iii) physicochemical and biological properties of P10Y complexes, and (iv) demonstrate their applicability as siRNA therapeutic in vivo (v) in the absence of adverse effects., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Polyethylenimines (PEIs), a group of polycationic molecules, are known to impair the outer membrane of Gram-negative bacteria and exhibit antimicrobial activity. The outer membrane of Gram-negative strains hinders the uptake of photosensitizer chlorophyllin. In this study, we report chlorophyllin and branched PEI combinations' activity against Escherichia coli strains DH5α and RB791, Salmonella enterica sv. Typhimurium LT2, and Bacillus subtilis 168. The minimal bactericidal concentration (MBC) was determined by plating cells treated with different concentrations of PEI and chlorophyllin on agar and monitoring their growth after 24 h. All tested combinations of PEI and chlorophyllin were lethal for S. enterica after 240 min of incubation in light, whereas PEI alone (<100 µg mL−1) was ineffective. In the darkness, complete inhibition was noted with a combination of ≥2.5 µg mL−1 chlorophyllin and 50 µg mL−1 PEI. If applied alone, PEI alone of ≥800 µg mL−1 of PEI was required to completely inactivate E. coli DH5α cells in light, whereas with ≥5 µg mL−1 chlorophyllin, only ≥100 µg mL−1 PEI was needed. No effect was detected in darkness with PEI alone. However, 1600 µg mL−1 PEI in combination with 2.5 µg mL−1 resulted in complete inactivation after 4 h dark incubation. PEI alone did not inhibit E. coli strain RB791, while cells were inactivated when treated with 10 µg mL−1 chlorophyllin in combination with ≥100 µg mL−1 (in light) or ≥800 µg mL−1 PEI (in darkness). Under illumination, B. subtilis was inactivated at all tested concentrations. In the darkness, 1 µg mL−1 chlorophyllin and 12.5 µg mL−1 PEI were lethal for B. subtilis. Overall, PEI can be used as an antimicrobial agent or potentiating agent for ameliorating the antimicrobial activity of chlorophyllin. [ABSTRACT FROM AUTHOR]