Endocytosis of Gene Delivery Vectors From Clathrin-dependent to Lipid Raft-mediated Endocytosis

Purpose: Nonviral gene delivery vectors remain less efficient than viral vectors, and their intracellular fate is strongly influenced by the endocytic route they enter. The cell membrane is now understood to contain lipid raft and non-raft domains, and the initial binding domain can determine the primary endocytic vesicle composition, associated regulators, and downstream trafficking. This review re-examines and reclassifies mammalian endocytic pathways in relation to lipid rafts, with emphasis on less-addressed lipid raft-mediated routes.

Hypothesis: The authors’ framing thesis: if a gene delivery vector binds to a specific plasma membrane domain—non-lipid raft, mixed, or lipid raft—then this determines the primary endocytic vesicle composition, the molecular regulators involved, and the subsequent intracellular itinerary; therefore, classifying pathways by membrane domain and targeting the appropriate route can improve nonviral gene delivery and endosomal escape.

Aims: Assess currently recognized endocytic pathways in mammalian cells. - Reclassify endocytic pathways based on the membrane regions that form the primary endocytic vesicles, especially in relation to lipid rafts. - Cover well-recognized pathways (clathrin-mediated endocytosis, phagocytosis, macropinocytosis) and less-addressed lipid raft pathways: caveolae-mediated, flotillin-dependent, GRAF1-dependent/CLIC-GEEC, Arf6-dependent, and RhoA-dependent endocytosis. - Summarize regulators, methods for interference/inhibition, and the fate of endocytic vesicles, and discuss implications for nonviral vector design.

Delivery system: General platform: nonviral gene delivery vectors that compact nucleic acids into nanosized particles. - Payloads: plasmid DNA, siRNA, anti-miRNA oligonucleotides, antisense oligonucleotides. - Vector examples discussed: lipoplexes, polyplexes, liposomes, PEI polyplexes, pDMAEMA polyplexes, polylysine-PEG/DNA polyplexes, bioreducible poly(amidoamine)/DNA polyplexes, PAMAM dendrimers, hydrophobically modified chitosan nanoparticles, amorphous silica nanoparticles, folate-modified branched PEI, c-NGR-targeted PLA-PEG nanoparticles, IRQ peptide-modified liposomes, and albumin-bound paclitaxel (Abraxane) as a transcytosis example. - Membrane/endocytic routes covered: CME; phagocytosis; macropinocytosis; caveolae-mediated endocytosis; flotillin-dependent endocytosis; GRAF1-dependent/CLIC-GEEC endocytosis; Arf6-dependent endocytosis; RhoA-dependent endocytosis. - Key regulators discussed: clathrin, dynamin-2, caveolin-1, cavins, flotillin-1/2, Fyn kinase, Cdc42, Arf1, GRAF1, Arf6, Rac1, RhoA, actin, cortactin, and PAK-1/2.

Approach: Review and synthesis of in vitro and in vivo literature. Cell models include HeLa, A431, COS-7, CHO, A549, HT1080, NIH 3T3, HepG2, B16-F10, HUVEC, brain microvessel endothelial cells, and others. In vivo examples include brain capillaries, lung endothelium, endothelial transcytosis, and tumor-related delivery. As a review, it reports no primary experimental groups, n values, doses, or controls.

Key methods: No primary methods. The review discusses data generated by cited studies using: - Total internal reflection fluorescence microscopy, confocal microscopy, electron microscopy, and dynamic colocalization microscopy. - Lipidomics and proteomics for membrane domain characterization. - RNAi knockdown of endocytic regulators. - Chemical inhibitors and cholesterol depletion. - Colocalization with pathway markers and fluid-phase uptake markers. - Transcytosis assays and in vivo biodistribution.

Key results: Reclassification: CME occurs in non-lipid raft domains; phagocytosis and macropinocytosis occur in mixed membrane domains; caveolae-, flotillin-, GRAF1-, Arf6-, and RhoA-dependent pathways occur in lipid raft domains. - Size ranges: clathrin-coated pits are ~150 nm but can reach or slightly exceed 200 nm; caveolae are 60–80 nm; macropinosomes can reach up to 10 µm; phagocytosis typically handles particles >0.5 µm. One study found lactosylated PEI polyplexes ≤100 nm accumulated in caveolae, 100–200 nm particles used CME, and >200 nm particles used macropinocytosis. - Caveolae: constitute 50–70% of lung endothelial cell surface membrane; a single caveola can internalize up to three 20 nm nanoparticles or two 40 nm nanoparticles; particles <40 nm were taken up 5–10 times more efficiently than 100 nm particles. - Fate: CME vesicles mature from early endosome (pH 6.1–6.8) to late endosome (pH 4.8–6) to lysosome (pH ~4.5). Caveolae in non-endothelial cells route to endolysosomes; in endothelial cells they can mediate transcytosis. Flotillin-positive vesicles route to late endosomes/lysosomes. GRAF1/CLIC-GEEC vesicles acquire Rab5/EEA1. Arf6-dependent cargo commonly recycles. RhoA-dependent IL2-R routes to early endosomes and then late endosomes/endolysosomes. - Inhibitor limitations: chlorpromazine, dynasore, cholesterol depletion, and amiloride are not pathway-specific; dynamin dependency is controversial for several pathways. GRAF1-dependent uptake was estimated to account for >70% of constitutive fluid uptake in NIH 3T3 cells.

Interpretation: The authors conclude that the endocytic pathway used by a nonviral vector strongly affects its intracellular fate. They suggest that caveolar endocytosis or macropinocytosis may allow more efficient endosomal escape than CME, because CME can lead to rapid lysosomal degradation. They argue that recognizing the membrane domain and specific regulators of each pathway will help avoid nonspecific inhibitors, better predict vector trafficking, and inspire more efficient virus-mimicking nonviral vectors.

Limitations: Review article; no primary data, systematic meta-analysis, effect sizes, n values, doses, or controls. - Dynamin dependency remains controversial for several pathways. - Selective chemical inhibitors are lacking for phagocytosis, macropinocytosis, flotillin-dependent, and GRAF1-dependent endocytosis. - Cholesterol depletion and other common inhibitors affect multiple pathways and can alter membrane organization. - Direct controlled studies linking a specific endocytic pathway to cytosolic escape and gene expression efficiency are limited. - Many lipid raft-mediated pathways remain less studied, and their exact intracellular fates are incompletely resolved.