Lipid nanoparticle-mediated messenger RNA delivery for ex vivo engineering of natural killer cells

Natural killer (NK) cells participate in the immune system by eliminating cancer and virally infected cells through germline-encoded surface receptors. Their independence from prior activation as well as their significantly lower toxicity have placed them in the spotlight as an alternative to T cells for adoptive cell therapy (ACT). Engineering NK cells with mRNA has shown great potential in ACT by enhancing their tumor targeting and cytotoxicity. However, mRNA transfection of NK cells is challenging, as the most common delivery methods, such as electroporation, show limitations. Therefore, an alternative non-viral delivery system that enables high mRNA transfection efficiency with preservation of the cell viability would be beneficial for the development of NK cell therapies. In this study, we investigated both polymeric and lipid nanoparticle (LNP) formulations for eGFP-mRNA delivery to NK cells, based on a dimethylethanolamine and diethylethanolamine polymeric library and on different ionizable lipids, respectively. The mRNA nanoparticles based on cationic polymers showed limited internalization by NK cells and low transfection efficiency. On the other hand, mRNA-LNP formulations were optimized by tailoring the lipid composition and the microfluidic parameters, resulting in a high transfection efficiency (∼100%) and high protein expression in NK cells. In conclusion, compared to polyplexes and electroporation, the optimized LNPs show a greater transfection efficiency and higher overall eGFP expression, when tested in NK (KHYG-1) and T (Jurkat) cell lines, and cord blood-derived NK cells. Thus, LNP-based mRNA delivery represents a promising strategy to further develop novel NK cell therapies

Nanoscopic structure of complexes formed between DNA and the cell-penetrating peptide penetratin

One of the most remarkable examples of cell-penetrating peptides (CPPs) is Penetratin, a 16-mer fragment derived from the Drosophila Antennapedia homeobox. Understanding the structure of Penetratin/DNA complexes is a key factor for the successful design of new vectors for gene delivery and may assist in optimizing molecular carriers based on CPPs. Herein, we present a comprehensive study on the nanoscale structure of noncovalent complexes formed between Penetratin and DNA. The strong cationic nature of the peptide makes it a very efficient agent for condensing DNA strands via electrostatic attraction, and we show for the first time that DNA condensation is accompanied by random-to-β-sheet transitions of Penetratin secondary structure, demonstrating that nucleic acids behave as a structuring agent upon complexation. For the first time, nanoscale-resolved spectroscopy is used to provide single-particle infrared data from DNA carriers based on CPPs, and they show that the structures are stabilized by Penetratin β-sheet cores, whereas larger DNA fractions are preferentially located in the periphery of aggregates. In-solution infrared assays indicate that phosphate diester groups are strongly affected upon DNA condensation, presumably as a consequence of charge delocalization induced by the proximity of cationic amide groups in Penetratin. The morphology is characterized by nanoassemblies with surface fractal features, and short-range order is found in the inner structure of the scaffolds. Interestingly, the formation of beads-on-a-string arrays is found, producing nanoscale architectures that resemble structures observed in early steps of chromatin condensation. A complexation pathway where DNA condensation and peptide pairing into β-sheets are key steps for organization is proposed

Desenvolvimento de nanopartículas de quitossomas como carreadores gênicos

Chitososomes are nanoparticles formed by interaction between liposomes and chitosan and have of particular interest for biotech applications due to their properties such as ease of production, low cost, storage stability, biodegradability and low immunogenicity and cytotoxicity. The production and use of chitosomes for several applications has been studied and is promising, because this nanoparticle is formed by a stable ternary complex. However, there are few studies in the literature of interaction of these nanoparticles with DNA, necessitating this study for the possible application of this nanoparticle as a gene carrier. Thus, the aim of this work was to evaluate the effect of arginine-modified chitosan on the formation of chitosomes and to study the interaction between different chitosomes with plasmid DNA pEGFP-N3. For this purpose, chitosan (CH) was modified with arginine (CH-Arg) and different chitosomes were synthesized by the reverse phase evaporation technique by the association of DOTAP/DOPE lipids with different amounts of CH-Arg. Chitosomes were complexed with pEGFP-N3 and their structures were evaluated by electrophoresis, zeta potential, dynamic light scattering, SAXS and isothermal titration calorimetry. In addition, the complexes were tested in vitro for evaluation of the transfection rate and cytotoxicity of the complexes. The results showed that the nanoparticles synthesized had a positive surface charge and average size of 116 nm and were able to complex with the pDNA through exothermic and thermodynamically favored interaction, and this complex remained positive enough not to suffer aggregation. The size, the positive charge of the complex and the stable interaction with the pDNA may have favored the entry of the nanoparticle into the cell by the electrostatic interaction with the membrane and the presence of CH-Arg in the compositions may have improved the efficiency of the endosomal escape, efficient carriers and non-cytotoxic complexes.Quitossomas são nanopartículas formadas pela interação entre lipossomas e quitosana e possuem especial interesse para aplicações biotecnológicas devido às suas propriedades, como facilidade de produção, baixo custo, estabilidade de armazenamento, biodegradabilidade e baixas imunogenicidade e citotoxicidade. A produção e a utilização de quitossomas para diversas aplicações já vem sendo estudada e mostra-se promissora, já que esta nanopartícula é formada por um complexo ternário estável. No entanto, há poucos estudos na literatura sobre a interação dessas nanopartículas com o DNA, necessitando desse estudo para a aplicação dessa nanopartícula como carreador gênico. Dessa forma, neste trabalho buscou-se avaliar o efeito da quitosana modificada com arginina na formação de quitossomas e estudar a interação entre diferentes quitossomas com o DNA plasmidial pEGFP-N3. Para tanto, realizou-se a modificação da quitosana com arginina (CH-Arg) e diferentes quitossomas foram sintetizados pela técnica de evaporação em fase reversa por meio da associação dos lipídeos DOTAP/DOPE com diferentes quantidades de CH-Arg. Os quitossomas foram complexados com o pEGFP-N3 e suas estruturas foram avaliadas pelas técnicas de eletroforese, potencial zeta, espalhamento de luz dinâmico, SAXS e calorimetria de titulação isotérmica. Além disso, os complexos foram testados in vitro para avaliação da taxa de transfecção e citotoxicidade dos complexos. Os resultados mostraram que as nanopartículas sintetizadas apresentam carga superficial positiva e tamanho médio de 116 nm e foram capazes de se complexarem com o pDNA através de interação exotérmica e termodinamicamente favorecida, sendo que este complexo manteve-se com um potencial positivo o suficiente para não sofrer agregação. O tamanho, a carga positiva do complexo e a interação estável com o pDNA podem ter favorecido a entrada da nanopartícula na célula pela interação eletrostática com a membrana plasmática e a presença de CH-Arg nas composições pode ter melhorado a eficiência do escape endossomal, tornando esses complexos eficientes carreadores e não citotóxicos.Dados abertos - Sucupira - Teses e dissertações (2019

Amyloid-like self-assembly of a hydrophobic cell-penetrating peptide and its use as a carrier for nucleic acids

Cell-penetrating peptides (CPPs) are a topic subject potentially exploitable for creating new nanotherapeutics for the delivery of bioactive loads. These compounds are often classified into three major categories according to their physicochemical characteristics: cationic, amphiphilic, and hydrophobic. Among them, the group of hydrophobic CPPs has received increasing attention in recent years due to toxicity concerns posed by highly cationic CPPs. The hexapeptide PFVYLI (P: proline, F: phenylalanine, V: valine, Y: tyrosine, L: leucine and I: isoleucine), a fragment derived from the C-terminal portion of α1-antitrypsin, is a prototypal example of hydrophobic CPP. This sequence shows reduced cytotoxicity, capacity of nuclear localization, and its small size readily hints suitability as a building block to construct nanostructured materials. In this study, we examine the self-assembling properties of PFVYLI and investigate its ability to form non-covalent complexes with nucleic acids. By using a combination of biophysical tools including synchrotron small-angle X-ray scattering and atomic force microscopy-based infrared spectroscopy, we discovered that this CPP self-assembles into discrete nanofibrils with remarkable amyloidogenic features. Over the course of days, these fibrils coalesce into rod-like crystals that easily reach the micrometer range. Despite lacking cationic residues in the composition, PFVYLI forms non-covalent complexes with nucleic acids that retain -sheet pairing found in amyloid aggregates. In vitro vectorization experiments performed with double-stranded DNA fragments indicate that complexes promote the internalization of nucleic acids, revealing that tropism toward cell membranes is preserved upon complexation. On the other hand, transfection assays with splice-correction oligonucleotides (SCOs) for luciferase expression show limited bioactivity across a narrow concentration window, suggesting that propensity to form amyloidogenic aggregates may trigger endosomal entrapment. We anticipate that the findings presented here open perspectives for using this archetypical hydrophobic CPP in the fabrication of nanostructured scaffolds, which potentially integrate properties of amyloids and translocation capabilities of CPPs.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-