Efficient Delivery of DNA Using Lipid Nanoparticles

Abstract

DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, the therapeutic efficacy of DNA vaccines has been limited by their intrinsic poor cellular internalization. Electroporation, which is based on the application of a controlled electric field to enhance DNA penetration into cells, has been the method of choice to produce acceptable levels of gene transfer in vivo. However, this method may cause cell damage or rupture, non-specific targeting, and even degradation of pDNA. Skin irritation, muscle contractions, pain, alterations in skin structure, and irreversible cell damage have been frequently reported. To overcome these limitations, in this work, we use a microfluidic platform to generate DNA-loaded lipid nanoparticles (LNPs) which are then characterized by a combination of dynamic light scattering (DLS), synchrotron small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Despite the clinical successes obtained by LNPs for mRNA and siRNA delivery, little is known about LNPs encapsulating bulkier DNA molecules, the clinical application of which remains challenging. For in vitro screening, LNPs were administered to human embryonic kidney 293 (HEK-293) and Chinese hamster ovary (CHO) cell lines and ranked for their transfection efficiency (TE) and cytotoxicity. The LNP formulation exhibiting the highest TE and the lowest cytotoxicity was then tested for the delivery of the DNA vaccine pVAX-hECTM targeting the human neoantigen HER2, an oncoprotein overexpressed in several cancer types. Using fluorescence-activated cell sorting (FACS), immunofluorescence assays and fluorescence confocal microscopy (FCS), we proved that pVAX-hECTM-loaded LNPs produce massive expression of the HER2 antigen on the cell membrane of HEK-293 cells. Our results provide new insights into the structure–activity relationship of DNA-loaded LNPs and pave the way for the access of this gene delivery technology to preclinical studies.

Protective efficacy induced by Eimeria maxima rhomboid-like protein 1 against homologous infection

“…Some non-injection routes are clinically popular, such as oral administration and spray. Therefore, the non-injection route could be used to evaluate the protective efficacy of EmROM1 to make it more promising for clinical application ( 52 , 53 ). Theoretically, the DNA vaccine is immunized once, and the organism is able to produce the target antigen continuously ( 52 ).…”

TGFβ signaling pathways in human health and disease

“…These nanoparticles are made of a polyethylene (PEG) lipid, phospholipid, cholesterol, and a cationic lipid and have an average particle size of 70–90 nm and are suitable for delivery of DNA and RNA molecules ( Brader et al, 2021 ; Cui et al, 2022 ). When loaded with RNA, LNPs not only protect their payload from degradation but are also capable of delivering it to cells and facilitate their intracellular entry ( Whitehead et al, 2009 ).…”

Formulation of Nucleic Acids by Encapsulation in Lipid Nanoparticles for Continuous Production of mRNA

“…LNP formulation for (p)DNA delivery has been reported in the literature, e.g., for LNP systems as transfection reagents for plasmid DNA (pDNA) [29], for enahnced oral delivery of DNA [30], for increased efficiency of DNA-vectored vaccines/immunoprophylaxis in animals [31], for spherical nucleic acids for intracellular DNA and RNA delivery [32], for transfection of cell model lines [33], as nanocarrier to deliver a cancer vaccine against the benchmark target tyrosine-kinase receptor HER2 in C57BL/6 mice [34], as well as in evaluating studies regarding the role of the influential Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery factors [26].…”