The steep road to nonviral nanomedicines: Frequent challenges and culprits in designing nanoparticles for gene therapy

Currently, extensive research is focused on the improvement of NP carrier design and gene delivery efficiency [23-26]. Recent debates within the gene delivery field have highlighted the need for an improved understanding about what cellular/subcellular features underscore the success of one formulation or the failure of another [15,17,27,28].

Within this context, an essential question is: “how are NPs internalized by cells?” [29]. Several mechanisms of endocytosis have been identified as listed in Table 2 [1]. Conventionally, the significance of an endocytosis pathway for a particular type of NP can be measured using pharmacological inhibitors or genetic approaches that knockdown/knockout or transiently block the expression of key proteins involved in endocytosis [30]. Then, changes in NP uptake can be quantified and attributed to the significance of that pathway. Unfortunately, upon limiting the function of a certain endocytosis pathway, especially through the use of pharmacological inhibitors, cells can respond to the blockage by overactivating alternative mechanisms that would normally be less relevant [31]. This makes the interpretation of each pathway’s significance more complicated and sometimes ambiguous [32]. To circumvent these issues, given the advancement and prevalence of high- or super-resolution microscopy, imaging-based approaches can be used to directly visualize uptake and determine whether the NP is co-localized or associated with key endocytic structures or proteins [33,34].

Table 2: Toolbox of pharmacological inhibitors used to study endocytosis pathways.

Pathways Commonly used pharmacological inhibitors Mechanism of inhibitors Potential pitfall of inhibitors Conc. range macropinocytosis amiloride and its derivatives 5-(N-ethyl-N-isopropyl) amiloride (EIPA) inhibits Na+ channels and Na+/H+ exchange (i) has been shown to inhibit FEMEa;
(ii) may affect actin
[37] 1 mM for amiloride and 25-100 μM for EIPA
[30,33] CMEb chloropromazine disrupts clathrin and the AP2 complex from the cell surface inhibits FEME, not efficient in all cell lines, affects biogenesis of large intracellular vesicles such as phagosomes and macropinosomes 30–60 μM
[30,38] caveolae methyl-β-cyclodextrin removes cholesterol from the plasma membrane (i) interferes with other uptake mechanisms [39] including macropinocytosis and CME because of changes in membrane fluidity;
(ii) affects actin cytoskeleton [40] 2.5–10 mM
[30,41,42]   filipin III interacts with cholesterol at the cell membrane (i) permeabilization of the plasma membrane occurs;
(ii) involves disruption of the linkages between F-actin and plasma membrane [35] 1–5 μg/mL
[43-46]

aFEME: clathrin-independent/dynamin-dependent endocytosis; bCME: clathrin-mediated endocytosis.

Overall, there is still a strong tendency for researchers to select pharmacological inhibition over genetic studies. This is due to several considerations, namely (i) rapid action in blocking the uptake route, (ii) equal inhibition of the overall cell population, and (iii) time- and labor-efficient processing [29,35]. However, it is well known that pharmacological inhibitors have severe limitations. These include varying levels of specificity (Table 2), significant cytotoxicity, low selectivity, and variable efficacy that can change within different cells lines and different experimental setups [1,30,35]. The proper use of transport inhibitors requires stringent controls to substantiate their effects and to rule out artifacts. Typically, this requires the use of appropriate markers that have been extensively validated to be specifically internalized by particular pathways. Transferrin [1] can be used as a maker for clathrin-mediated endocytosis (CME), bodipy-lactosylceramide (LacCer) can be used for caveolae-mediated endocytosis, and dextran with large molecular masses can be used for macropinocytosis [36]. Moreover, concentration should be optimized (Table 2) and toxicity should be closely monitored, as cell death can be misinterpreted as efficient inhibition, especially in metabolism-based assays. For these inhibitors that involve the permeablization of plasma membranes, such as filipin III, appropriate controls for plasma membrane integrity during the inhibition exposure should be included [35].

In order to overcome the poor specificity of pharmacological inhibitors, genetic approaches can be implemented to change the expression of specific proteins [29]. However, the complexity and care with which these studies must be undertaken are higher than an inhibition approach. One genetic approach is the knockout/knockdown of key components in internalization pathways [47]. Alternatively, the expression of dominant-negative inhibitors may be used [48]. If performed carefully with appropriate controls, both can largely overcome major problems associated with pharmacological inhibitors. However, genetic alterations may also result in changes that share protein components or lead to compensatory mechanisms in the cell [37]. Despite their intrinsic specificity, validation still remains critical to avoid affecting multiple pathways. Hence, the inclusion of appropriate positive and negative controls is crucial to avoid misinterpretations if genetic approaches are used.

In addition to (or in tandem with) inhibition and genetic strategies, imaging-based approaches should be performed. These imaging studies aid in the identification of the internalization mechanisms via fluorescence co-localization analysis of endocytic biological markers and NPs [33]. While co-localization studies by means of overexpression of fluorescent proteins of key endocytic regulators or immunostaining for these regulators can be helpful in determining endocytosis routes of NP uptake, care should be taken to prevent artifacts [49]. New biological tools, such as SNAP-tag, could be used to label intracellular proteins with high efficiency and low fluorescence background, which would be promising for future co-localization studies investigating NP-mediated endocytic routes [50,51].

The combination of multiple methods among those available (each presenting advantages and limits) is probably the best approach to try to fully characterize and investigate the pathways involved and answer essential questions. An idealized scenario may be one in which inhibition results are coupled to both knockdown and imaging data to provide a full understanding of the mode of action in NP uptake studies.

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