Cancer is an extremely complex disease, typically caused by mutations in cancer-critical genes. By delivering therapeutic nucleic acids (NAs) to patients, gene therapy offers the possibility to supplement, repair or silence such faulty genes or to stimulate their immune system to fight the disease. While the challenges of gene therapy for cancer are significant, the latter approach (a type of immunotherapy) starts showing promising results in early-stage clinical trials. One important advantage of NA-based cancer therapies over synthetic drugs and protein treatments is the prospect of a more universal approach to designing therapies. Designing NAs with different sequences, for different targets, can be achieved by using the same technologies. This versatility and scalability of NA drug design and production on demand open the way for more efficient, affordable and personalized cancer treatments in the future. However, the delivery of exogenous therapeutic NAs into the patients’ targeted cells is also challenging. Membrane-type lipids exhibiting permanent or transient cationic character have been shown to associate with NAs (anionic), forming nanosized lipid-NA complexes. These complexes form a wide variety of nanostructures, depending on the global formulation composition and properties of the lipids and NAs. Importantly, these different lipid-NA nanostructures interact with cells via different mechanisms and their therapeutic potential can be optimized to promising levels in vitro. The complexes are also highly customizable in terms of surface charge and functionalization to allow a wide range of targeting and smart-release properties. Most importantly, these synthetic particles offer possibilities for scaling-up and affordability for the population at large. Hence, the versatility and scalability of these particles seem ideal to accommodate the versatility that NA therapies offer. While in vivo efficiency of lipid-NA complexes is still poor in most cases, the advances achieved in the last three decades are significant and very recently a lipid-based gene therapy medicine was approved for the first time (for treatment of hereditary transthyretin amyloidosis). Although the path to achieve efficient NA-delivery in cancer therapy is still long and tenuous, these advances set a new hope for more treatments in the future. In this review, we attempt to cover the most important biophysical and physicochemical aspects of non-viral lipid-based gene therapy formulations, with a perspective on future cancer treatments in mind.

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.

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Hybrid lipid-polymer-nucleic acid nanoparticles (LPNPs) provide a range of delivery strategies for nonviral gene therapy. However, due to several pairwise interactions between their components, these formulations are difficult to predict and characterize. Here, we employed, for the first time, a novel methodology based on fluorescence cross correlation spectroscopy (FCCS) to directly quantify the extent of association between polycation-DNA cores (polyplexes) and cationic liposome shells. This leads to a rapid and easy evaluation of LPNP formation. As a result, we were able to unveil two critical insights for predicting LPNP assembly. Firstly, the interaction between the polycation (polylysine) and DNA is robust, with the polycation not being displaced by liposomes. Hence, the polyplex cores and liposome shells have to be oppositely charged to associate. Secondly, and most importantly, the liposome:poliplex number ratio (ρN), as opposed to the more common charge ratio, is a critical parameter to predict stable LPNP formation. We find that ρN≥1 is required to ensure that every polyplex is enveloped by a liposome, avoiding the coexistence of oppositely charged species, thus inhibiting aggregation. Since the chosen LPNP components are common, these observations should be applicable to many LPNP systems. Furthermore, this FCCS-based methodology is relevant to other self-assembly composite materials, for applications beyond gene delivery.

Hybrid core–shell lipid–polycation–nucleic acid nanoparticles (LPNPs) provide unique delivery strategies for nonviral gene therapeutics. Since LPNPs consist of multiple components, involving different pairwise interactions between them, they are challenging to characterize and understand. Here, we propose a method based on fluorescence cross-correlation spectroscopy to elucidate the association between the three LPNP components. Through this lens, we demonstrate that cationic lipid shells (liposomes) do not displace polycations or DNA from the polycation–DNA cores (polyplexes). Hence, polyplexes and liposomes must be oppositely charged to associate into LPNPs. Furthermore, we identify the liposome:polyplex number ratio (ρ N ), which was hitherto an intangible quantity, as the primary parameter predicting stable LPNPs. We establish that ρ N ≥ 1 ensures that every polyplex is enveloped by a liposome, thus avoiding coexisting oppositely charged species prone to aggregation.