Efficient Transfection via an Unexpected Mechanism by Near Neutral Polypiperazines with Tailored Response to Endosomal pH

Hausig‐Punke, Franziska; Dekevic, Gregor; Sobotta, Fabian H.; Solomun, Jana I.; Richter, Friederike; Salzig, Denise; Traeger, Anja; Brendel, Johannes C.

doi:10.1002/mabi.202370016

Cited by 3 publications

(2 citation statements)

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“…The application of cationic pH-responsive polymers, the polypiperazines, is also promising. These avoid the cytotoxic effect of lPEI and represent a new hydrophilic class of pH-responsive polymers, whose response can be tuned within the relevant pH 5–7.4 with high transfection efficiencies [ 109 ].…”

Section: Discussionmentioning

confidence: 99%

A Bioreactor-Based Yellow Fever Virus-like Particle Production Process with Integrated Process Analytical Technology Based on Transient Transfection

Dekevic,

Tertel,

Tasto

et al. 2023

Viruses

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Yellow Fever (YF) is a severe disease that, while preventable through vaccination, lacks rapid intervention options for those already infected. There is an urgent need for passive immunization techniques using YF-virus-like particles (YF-VLPs). To address this, we successfully established a bioreactor-based production process for YF-VLPs, leveraging transient transfection and integrating Process Analytical Technology. A cornerstone of this approach was the optimization of plasmid DNA (pDNA) production to a yield of 11 mg/L using design of experiments. Glucose, NaCl, yeast extract, and a phosphate buffer showed significant influence on specific pDNA yield. The preliminary work for VLP-production in bioreactor showed adjustments to the HEK cell density, the polyplex formation duration, and medium exchanges effectively elevated transfection efficiencies. The additive Pluronic F-68 was neutral in its effects, and anti-clumping agents (ACA) adversely affected the transfection process. Finally, we established the stirred-tank bioreactor process with integrated dielectric spectroscopy, which gave real-time insight in relevant process steps, e.g., cell growth, polyplex uptake, and harvest time. We confirmed the presence and integrity of YF-VLP via Western blot, imaging flow cytometry measurement, and transmission electron microscopy. The YF-VLP production process can serve as a platform to produce VLPs as passive immunizing agents against other neglected tropical diseases.

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Section: Discussionmentioning

confidence: 99%

A Bioreactor-Based Yellow Fever Virus-like Particle Production Process with Integrated Process Analytical Technology Based on Transient Transfection

Dekevic,

Tertel,

Tasto

et al. 2023

Viruses

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“…Another objective of our study was to explore various buffers and systematically see if the ion strength will influence the efficacy of siRNA polyplexes. Different solutions such as HEPES [43,44], water [45], PIPES buffer (20 mM PIPES, 150 mM NaCl) [46], HBG buffer (20 mm HEPES, 5% w/v glucose, pH 7.4) [47] and Tris-EDTA [48] have been investigated for nucleic acid complexation. Within this framework, we experimented with water, citrate and phosphate buffers and DMEM as the complexation media, with Ca 2+ as a special additive due to the previously observed benefit of this ion on nucleic acid complexes.…”

Section: Discussionmentioning

confidence: 99%

Designing Nanomedicines for Breast Cancer Therapy

Abbasi Dezfouli,

Rajendran,

Claerhout

et al. 2023

Biomolecules

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In 2020, breast cancer became the most diagnosed cancer worldwide. Conventional chemotherapies have major side effects due to their non-specific activities. Alternatively, short interfering RNA(siRNA)-carrying nanoparticles (NPs) have a high potential to overcome this non-specificity. Lipid-substituted polyethyleneimine (PEI) polymers (lipopolymers) have been reported as efficient non-viral carriers of siRNA. This study aims to engineer novel siRNA/lipopolymer nanocomplexes by incorporating anionic additives to obtain gene silencing through siRNA activity with minimal nonspecific toxicity. We first optimized our polyplexes in GFP+ MDA-MB-231 cells to effectively silence the GFP gene. Inclusion of phosphate buffer with pH 8.0 as complex preparation media and N-Lauroylsarcosine Sodium Salt as additive, achieved ~80% silencing with the least amount of undesired cytotoxicity, which was persistent for at least 6 days. The survivin gene was then selected as a target in MDA-MB-231 cells since there is no strong drug (i.e., small organic molecule) for inhibition of its oncogenic activity. The qRT-PCR, flow cytometry analysis and MTT assay revealed >80% silencing, ~95% cell uptake and >70% cell killing by the same formulation. We conclude that our lipopolymer can be further investigated as a lead non-viral carrier for breast cancer gene therapy.

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Polypiperazine-Based Micelles of Mixed Composition for Gene Delivery

Stancheva,

Haladjova,

Petrova

et al. 2024

Polymers

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We introduce a novel concept in nucleic acid delivery based on the use of mixed polymeric micelles (MPMs) as platforms for the preparation of micelleplexes with DNA. MPMs were prepared by the co-assembly of a cationic copolymer, poly(1-(4-methylpiperazin-1-yl)-propenone)-b-poly(d,l-lactide), and nonionic poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) block copolymers. We hypothesize that by introducing nonionic entities incorporated into the mixed co-assembled structures, the mode and strength of DNA binding and DNA accessibility and release could be modulated. The systems were characterized in terms of size, surface potential, buffering capacity, and binding ability to investigate the influence of composition, in particular, the poly(ethylene oxide) chain length on the properties and structure of the MPMs. Endo–lysosomal conditions were simulated to follow the changes in fundamental parameters and behavior of the micelleplexes. The results were interpreted as reflecting the specific structure and composition of the corona and localization of DNA in the corona, predetermined by the poly(ethylene oxide) chain length. A favorable effect of the introduction of the nonionic block copolymer component in the MPMs and micelleplexes thereof was the enhancement of biocompatibility. The slight reduction of the transfection efficiency of the MPM-based micelleplexes compared to that of the single-component polymer micelles was attributed to the premature release of DNA from the MPM-based micelleplexes in the endo–lysosomal compartments.

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Efficient Transfection via an Unexpected Mechanism by Near Neutral Polypiperazines with Tailored Response to Endosomal pH

Cited by 3 publications

References 0 publications

A Bioreactor-Based Yellow Fever Virus-like Particle Production Process with Integrated Process Analytical Technology Based on Transient Transfection

A Bioreactor-Based Yellow Fever Virus-like Particle Production Process with Integrated Process Analytical Technology Based on Transient Transfection

Designing Nanomedicines for Breast Cancer Therapy

Polypiperazine-Based Micelles of Mixed Composition for Gene Delivery

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