The approval of Onpattro® (2018) and Comirnaty (2020) has driven interest in nanoparticulate nucleotide delivery. Newer concepts in gene therapy however, require not only the delivery of one, but multiple nucleotides. Examples are CRISPR/Cas9 gene editing and cancer immunotherapy. However, the current gold standard for nucleotide delivery - lipid nanoparticles - faces significant challenges, including limitations for co-encapsulation and nucleotide-nucleotide interactions. Aim of this study was to design a core-shell system featuring separate encapsulation of two nucleotides via a two-step formulation process. Six distinct cationic polymers were combined with three anionic polymers, resulting in 18 core compositions. Screening of these formulations identified three potent lipopolyplexes (LPPs), which were further evaluated and compared in terms of transfection efficiency, expression kinetics, storage stability, and nebulization performance. Among them, the combination of poly-L-arginine and poly-L-glutamic acid demonstrated the highest overall performance. Our systems enabled precise co-delivery of two model mRNAs in a controlled ratio, demonstrating potential for advanced therapeutic applications. Additionally, the role of mRNA localization within the LPP was investigated. Surface-loaded mRNA demonstrated superior transfection efficiency and shear resistance compared to core-loaded mRNA, which lost functionality under nebulization.

The lack of understanding of polyplexes stability and their dissociation mechanisms, allowing the release of DNA, is currently a major limitation in non-viral gene delivery. One proposed mechanism for DNA-based polyplexes dissociation is based on the electrostatic interactions between polycations and biological polyanions, such as glycosaminoglycans (GAGs). This work aimed at investigating whether GAGs such as heparin, chondroitin sulphate and hyaluronic acid promote the dissociation of PEI/DNA polyplexes. We studied the electrostatic complexation between branched poly(ethyleneimine) (b-PEI25) and polyanions (model DNA and GAGs) through conductivity and ζ-potential measurements. The formation of b-PEI25/polyanion polyplexes through electrostatic interactions was analyzed in depth, providing key insights into charge stoichiometry, morphology, thermodynamics and physicochemical characteristics. The stability of polyplexes was tested in the presence of the different GAGs. Heparin was found to be the only polyanion capable of releasing peGFP-C3 plasmid from polyplexes, complexing stoichiometrically with the free b-PEI25 in excess, before releasing the plasmid. The ability of GAGs to disrupt polyplexes and release DNA was correlated with the thermodynamic characteristics of b-PEI25/polyanions complexation. Our findings indicate that heparin's strong interaction with PEI and its high charge density, compared to other GAGs and polyanions, are pivotal in determining complex stability and promoting DNA release.

Cationic polymers are known to efficiently deliver nucleic acids to target cells by encapsulating the cargo into nanoparticles. However, the molecular organization of these nanoparticles is often not fully explored. Yet, this information is crucial to understand complex particle systems and the role influencing factors play at later stages of drug development. Coarse-grained molecular dynamics (CG-MD) enables modeling of systems that are the size of real nanoparticles, providing meaningful insights into molecular interactions between polymers and nucleic acids. Herein, the particle assembly of variations of an amphiphilic poly(beta-amino ester) (PBAE) with siRNA was simulated to investigate the influence of factors such as polymer lipophilicity and buffer conditions on the nanoparticle structure. Simulations were validated by wet lab methods including nuclear magnetic resonance (NMR) and align well with experimental findings. Therefore, this work emphasizes that CG-MD simulations can provide underlying explanations of experimentally observed nanoparticle properties by visualizing the nanoscale structure of polyplexes.

The objective of this study is to develop a versatile gene carrier based on lipopeptides capable of delivering genetic material into target cells with minimal cytotoxicity.

Two lipopeptide molecules, palmitoyl-CKKHH and palmitoyl-CKKHH-YGRKKRRQRRR-PKKKRKV, were synthesized using solid phase peptide synthesis and evaluated as transfection agents. Physicochemical characterization of the lipopeptides included a DNA shift mobility assay, particle size measurement, and transmission electron microscopy (TEM) analysis. Cytotoxicity was assessed in CHO-K1 and HepG2 cells using the MTT assay, while transfection efficiency was determined by evaluating the expression of the green fluorescent protein-encoding gene.

Our findings demonstrate that the lipopeptides can bind, condense, and shield DNA from DNase degradation. The inclusion of the YGRKKRRQRRR sequence, a transcription trans activator, and the PKKKRKV sequence, a nuclear localization signal, imparts desirable properties. Lipopeptide-based TAT-NLS/DNA nanoparticles exhibited stability for up to 20 days when stored at 6-8 °C, displaying uniformity with a compact size of approximately 120 nm. Furthermore, the lipopeptides exhibited lower cytotoxicity compared to the poly-L-lysine. Transfection experiments revealed that protein expression mediated by the lipopeptide occurred at a charge ratio ranging from 4.0 to 8.0.

These results indicate that the lipopeptide, composed of a palmitoyl alkyl chain and TAT and NLS sequences, can efficiently condense and protect DNA, form stable and uniform nanoparticles, and exhibit promising characteristics as a potential gene carrier with minimal cytotoxicity.