Choosing an Optimal Solvent Is Crucial for Obtaining Cell-Penetrating Peptide Nanoparticles with Desired Properties and High Activity in Nucleic Acid Delivery.
作者: Abhijit Biswas ; Maria Maloverjan ; Kärt Padari ; Aare Abroi ; Margus Rätsep ; Sebastian K T S Wärmländer ; Jüri Jarvet ; Astrid Gräslund ; Vambola Kisand ; Rünno Lõhmus ; Margus Pooga
Cell-penetrating peptides (CPPs) are highly promising transfection agents that can deliver various compounds into living cells, including nucleic acids (NAs). Positively charged CPPs can form non-covalent complexes with negatively charged NAs, enabling simple and time-efficient nanoparticle preparation. However, as CPPs have substantially different chemical and physical properties, their complexation with the cargo and characteristics of the resulting nanoparticles largely depends on the properties of the surrounding environment, i.e., solution. Here, we show that the solvent used for the initial dissolving of a CPP determines the properties of the resulting CPP particles formed in an aqueous solution, including the activity and toxicity of the CPP-NA complexes. Using different biophysical methods such as dynamic light scattering (DLS), atomic force microscopy (AFM), transmission and scanning electron microscopy (TEM and SEM), we show that PepFect14 (PF14), a cationic amphipathic CPP, forms spherical particles of uniform size when dissolved in organic solvents, such as ethanol and DMSO. Water-dissolved PF14, however, tends to form micelles and non-uniform aggregates. When dissolved in organic solvents, PF14 retains its α-helical conformation and biological activity in cell culture conditions without any increase in cytotoxicity. Altogether, our results indicate that by using a solvent that matches the chemical nature of the CPP, the properties of the peptide-cargo particles can be tuned in the desired way. This can be of critical importance for in vivo applications, where CPP particles that are too large, non-uniform, or prone to aggregation may induce severe consequences.
2019-12-27·Scientific reports3区 · 综合性期刊
Effective lung-targeted RNAi in mice with peptide-based delivery of nucleic acid.
3区 · 综合性期刊
作者: Kaido Kurrikoff ; Krista Freimann ; Kadi-Liis Veiman ; Elin Madli Peets ; Andres Piirsoo ; Ülo Langel
We have previously developed efficient peptide-based nucleic acid delivery vectors PF14 and NF55, where we have shown that these vectors preferentially transfect lung tissue upon systemic administration with the nucleic acid. In the current work, we have explored the utilization and potential of these vectors for the lung-targeted gene therapy. Accordingly, we assessed the efficacy of these peptides in (i) two different lung disease models - acute lung inflammation and asthma in mice and (ii) using two different nucleic acid cargos - siRNA and pDNA encoding shRNA. Using RNAi against cytokine TNFα, we showed efficient anti-inflammatory effects in both disease models and observed decreased disease symptoms. Our results highlight the potential of our transfection vectors for lung gene therapy.
2019-10-01·FASEB journal : official publication of the Federation of American Societies for Experimental Biology2区 · 生物学
The nuclear concentration required for antisense oligonucleotide activity in myotonic dystrophy cells.
2区 · 生物学
作者: M Leontien van der Bent ; Omar Paulino da Silva Filho ; Marieke Willemse ; Mattias Hällbrink ; Derick G Wansink ; Roland Brock
Antisense oligonucleotides (ASOs) are a promising class of therapeutics that are starting to emerge in the clinic. Determination of intracellular concentrations required for biologic effects and identification of effective delivery vehicles are crucial for understanding the mode of action and required dosing. Here, we investigated which nuclear oligonucleotide concentration is needed for a therapeutic effect for a triplet repeat-targeting ASO in a muscle cell model of myotonic dystrophy type 1 (DM1). For cellular delivery, ASOs were complexed into nanoparticles using the cationic cell-penetrating peptides nona-arginine and PepFect14 (PF14). Although both peptides facilitated uptake, only PF14 led to a dose-dependent correction of disease-typical abnormal splicing. In line with this observation, time-lapse confocal microscopy demonstrated that only PF14 mediated translocation of the ASOs to the nucleus, which is the main site of action. Through fluorescence lifetime imaging, we could distinguish intact oligonucleotide from free fluorophore, showing that PF14 also shielded the ASOs from degradation. Finally, we employed a combination of live-cell fluorescence correlation spectroscopy and immunofluorescence microscopy and demonstrated that intranuclear blocking-type oligonucleotide concentrations in the upper nanomolar range were required to dissolve nuclear muscleblind-like protein 1 foci, a hallmark of DM1. Our findings have important implications for the clinical use of ASOs in DM1 and provide a basis for further research on other types of ASOs.-Van der Bent, M. L., Paulino da Silva Filho, O., Willemse, M., Hällbrink, M., Wansink, D. G., Brock, R. The nuclear concentration required for antisense oligonucleotide activity in myotonic dystrophy cells.