In Vitro Evaluation of Lipopolyplexes for Gene Transfection: Comparing 2D, 3D and Microdroplet-Enabled Cell Culture

Abstract

Complexes combining nucleic acids with lipids and polymers (lipopolyplexes) show great promise for gene therapy since they enable compositional, physical and functional versatility to be optimized for therapeutic efficiency. When developing lipopolyplexes for gene delivery, one of the first evaluations performed is an in vitro transfection efficiency experiment. Many different in vitro models can be used, and the effect of the model on the experiment outcome has not been thoroughly studied. The objective of this work was to compare the insights obtained from three different in vitro models, as well as the potential limitations associated with each of them. We have prepared a series of lipopolyplex formulations with three different cationic polymers (poly-l-lysine, bioreducible poly-l-lysine and polyethyleneimine), and assessed their in vitro biological performance in 2D monolayer cell culture, 3D spheroid culture and microdroplet-based single-cell culture. Lipopolyplexes from different polymers presented varying degrees of transfection efficiency in all models. The best-performing formulation in 2D culture was the polyethyleneimine lipopolyplex, while lipoplexes prepared with bioreducible poly-l-lysine were the only ones achieving any transfection in microdroplet-enabled cell culture. None of the prepared formulations achieved significant gene transfection in 3D culture. All of the prepared formulations were well tolerated by cells in 2D culture, while at least one formulation (poly-l-lysine polyplex) delayed 3D spheroid growth. These results highlight the need for selecting the appropriate in vitro model depending on the intended application.

Endostatin Genetically Engineered Placental Mesenchymal Stromal Cells Carrying Doxorubicin-Loaded Mesoporous Silica Nanoparticles for Combined Chemo- and Antiangiogenic Therapy

“…To test the in vitro efficacy of the combined therapeutic strategy (cell platform), we employed a multicellular 3D spheroid model formed by the coculture of tumoral and endothelial cells. It is considered that spheroids mimic tumor behavior more accurately than two dimensional (2D) cell cultures and represent a much more relevant model for evaluating anticancer therapies [ 45 , 46 , 47 ]. Our multicellular model may provide information about the effect of the developed cell platform on cancer and endothelial cells simultaneously.…”

New Applications of Lipid and Polymer-Based Nanoparticles for Nucleic Acids Delivery

“…A recently approached strategy for designing performant hybrid carriers is the creation of lipopolyplexes (LPPs), complexes combining nucleic acids with lipids and polymers (Figure 5). Such structures are promising delivery systems for gene therapy, especially due to their compositional, physical, and functional versatility [131][132][133]. For instance, Wang et al [134] have synthesized a tumor-selective LPP consisting of a PEI/p21-saRNA-322 core and a hyaluronan-modulated lipid shell.…”

Dendrimers as Non-Viral Vectors in Gene-Directed Enzyme Prodrug Therapy

“…There are several types of nVVs, commonly used in GDEPT, such as: polyplexes - short-lived electrostatic complexes that require an excess of polymer [ 69 ]; dendriplexes [ 70 ], lipoplexes [ 71 ], lipopolyplexes [ 72 ], lypodendriplexes [ 73 ], and micelles - dynamic amphiphilic polymers [ 74 ]. Nano-systems include: mesoporous nanoparticles [ 75 , 76 ], exosomes / microvesicles [ 77 ], organic / inorganic hybrids [ 78 , 79 ], nanofibres for from natural polymers [ 80 , 81 ], elecrospun nanofibers [ 82 ], niosomes / nioplexes [ 83 ], etc.…”