Cellular Uptake of Nanoparticles: Journey Inside the Cell

Purpose: Nanoparticles (NPs) are increasingly used in consumer goods, electronics, and pharmaceuticals, but their beneficial and/or deleterious effects ultimately arise from interactions at the cellular and subcellular level. While many NPs require safe entry into cells for therapeutic efficacy, efficient and controlled entry/trafficking remains a major challenge. A comprehensive understanding of NP cellular uptake and trafficking mechanisms is critical for designing efficient and safe nanomedicines. ---

Hypothesis: The review’s central thesis is: if the physicochemical properties of NPs (size, shape, surface charge, surface functionality) and the biological context (cell type, microenvironment, protein corona) are systematically understood and tuned, then cellular uptake, intracellular trafficking, and therapeutic efficacy of NPs can be optimized while minimizing toxicity. ---

Aims: - Provide an objective and comprehensive account of current understanding of NP cellular uptake. - Discuss the underlying parameters controlling nano–cellular interactions, including NP physicochemical properties and biological factors. - Review the available analytical techniques to follow and track NP cellular uptake and trafficking processes. - Discuss intracellular trafficking, exocytosis, and artifacts/crucial “ignored” parameters in cellular uptake evaluation. ---

Delivery system: Nanoparticle types reviewed: - Polymeric NPs: PLGA, PLA, PEG-co-PLA, polyalkylcyanoacrylate, polystyrene, chitosan, alginate-chitosan, poly(methyl methacrylate)-block-PEG, N-acetyl histidine-conjugated glycol chitosan - Lipid-based: liposomes (Doxil), lipid nanoparticles (LNPs), solid lipid NPs - Inorganic: gold NPs (spheres, rods, cages), silica NPs (spherical, rod-shaped, mesoporous), quantum dots (CdTe, CdSe/ZnS), iron oxide (SPIONs), silver NPs, copper oxide NPs, carbon nanotubes, diamond NPs, (mathrm{Cu_3BiS_3}) nanodots - Protein-based: albumin-bound paclitaxel (Abraxane) Payloads: Doxorubicin, paclitaxel, siRNA, plasmid DNA, mRNA, sgRNA, contrast agents Targeting ligands: Transferrin, folate, aptamers, peptides, antibodies (Herceptin), affibodies Entry mechanisms covered: - Phagocytosis - Clathrin-mediated endocytosis (CME) - Caveolae-dependent endocytosis - Clathrin/caveolae-independent endocytosis - Macropinocytosis - Non-endocytic mechanisms: passive diffusion, hole formation, microinjection, electroporation ---

Approach: Narrative review of published literature. Model systems discussed include: - In vitro cell lines: HeLa, A549, MCF-7, MDA-MB-231, MDA-MB-435, HepG2, J774A.1, RAW 264.7, THP-1, 1321N1, NIH3T3, COS-7, CHO, HUVEC, BOEC, HMEC, PC-12, SK-BR-3, Caco-2, and many others. - In vivo models: Mouse models (C57BL/6, tumor-bearing mice), rat models. - Disease context: Cancer, diabetes, leishmaniasis, infectious diseases, inflammatory diseases. - Microenvironment considerations: Tumor microenvironment (TME), gastrointestinal tract, blood, cell-culture media. ---

Key methods: Techniques highlighted across cited studies: - Super-resolution fluorescence microscopy: STORM, PALM, confocal laser scanning microscopy (CLSM) - Transmission electron microscopy (TEM): ultrastructure, size/location, elemental composition - Atomic force microscopy (AFM): topographical and mechanical information - Scanning electron microscopy (SEM): surface imaging, FIB-SEM for tomography - Light-scattering microscopy: Raman microscopy, SERS - Flow cytometry: single-cell analysis, FACS, FRET - Dark-field microscopy: plasmonic NP tracking - Photoacoustic microscopy: individual cell imaging - Laser ablation ICP-MS: quantitative spatial distribution maps - X-ray adsorption near-edge spectroscopy (XANES): chemical fate/speciation - Correlative microscopy: integration of multiple techniques ---

Key results: - Size: ~50 nm is the optimum size for highest cellular uptake in certain cells; larger particles (1000–2000 nm) show maximal phagocytic uptake; smaller NPs (2.1 nm) can enter the nucleus/nucleoli, while 4.4 nm NPs show reduced penetration. - Shape: Spherical NPs undergo higher cellular uptake than rod-shaped NPs; lower aspect ratio rods are taken up faster than higher aspect ratio rods; rod and disc NPs are taken up twice as quickly as spheres by Caco-2 cells. - Surface charge: Cationic Au NPs show >5-fold greater uptake than anionic counterparts; positively charged NPs induce membrane depolarization and increased intracellular (mathrm{Ca^{2+}}); cationic NPs can directly induce hole formation in lipid bilayers. - Aggregation: Uptake of aggregated NPs is on average 25% lower than non-aggregated Au NPs for HeLa and A549 cells. - Protein corona: What cells “see” is corona-coated NPs, not pristine surfaces; the biological identity of NPs is determined by NP-related, biological, and experimental factors. - Exocytosis: Smaller NPs are more easily cleared from cells; rod-shaped NPs are excreted more than spherical NPs; lysosomal exocytosis plays a key role. - Cell type: Different cell types employ different endocytotic pathways for the same NP; caveolae-dependent endocytosis operates over one-third of the cell membrane in some tissues. ---

Interpretation: The authors conclude that although knowledge regarding cellular association and trafficking of nanoscale materials has advanced tremendously, much progress remains in understanding and exploiting context-dependent phenomena in various disease states. Advances in organ-on-a-chip technologies, subcellular analysis (super-resolution microscopy, cryo-electron microscopy, high-content analytics, imaging cytometry), and chemical synthesis will continue to expand the ability to exploit and treat human diseases using synthetic nanomaterials. ---

Limitations: - Artifacts in experiments: Toxicity may originate from supernatant agents (e.g., CTAB in gold nanorod preparations) rather than NPs themselves. - Colloidal stability: NPs stable in original solution may aggregate in biological media, altering uptake extent, rate, mechanism, and toxicity. - ICP-MS limitations: Cannot differentiate between internalized NPs and those attached to the cell surface. - “Cell vision” and “protein corona”: These hidden factors substantially change intracellular trafficking and complicate in vitro–in vivo extrapolation. - Disease-specific protein corona: The type of disease may change NP biological identity and fate. - Sedimentation vs. diffusion: In typical in vitro studies, aggregation may mask Brownian movement, with larger/heavier particles sedimenting first, leading to variability in dose available on the cell surface. - Lack of consensus: Scientists do not yet agree on the final destiny of NPs captured in macropinosomes. - As a review: Not a systematic review or meta-analysis; no primary data.