Clathrin to Lipid Raft-Endocytosis via Controlled Surface Chemistry and Efficient Perinuclear Targeting of Nanoparticle

Abstract

Nanoparticle interacts with live cells depending on their surface chemistry, enters into cell via endocytosis, and is commonly trafficked to an endosome/lysozome that restricts subcellular targeting options. Here we show that nanoparticle surface chemistry can be tuned to alter their cell uptake mechanism and subcellular trafficking. Quantum dot based nanoprobes of 20-30 nm hydrodynamic diameters have been synthesized with tunable surface charge (between +15 mV to -25 mV) and lipophilicity to influence their cellular uptake processes and subcellular trafficking. It is observed that cationic nanoprobe electrostatically interacts with cell membrane and enters into cell via clathrin-mediated endocytosis. At lower surface charge (between +10 mV to -10 mV), the electrostatic interaction with cell membrane becomes weaker, and additional lipid raft endocytosis is initiated. If a lipophilic functional group is introduced on a weakly anionic nanoparticle surface, the uptake mechanism shifts to predominant lipid raft-mediated endocytosis. In particular, the zwitterionic-lipophilic nanoprobe has the unique advantage as it weakly interacts with anionic cell membrane, migrates toward lipid rafts for interaction through lipophilic functional group, and induces lipid raft-mediated endocytosis. While predominate or partial clathrin-mediated entry traffics most of the nanoprobes to lysozome, predominate lipid raft-mediated entry traffics them to perinuclear region, particularly to the Golgi apparatus. This finding would guide in designing appropriate nanoprobe for subcellular targeting and delivery.

Preferential interactions of primary amine-terminated quantum dots with membrane domain boundaries and lipid rafts revealed with nanometer resolution

“…The ζ-potential of quantum dots has been suggested to dictate their route of entry into cells. 55 It has been shown that quantum dots with lower surface potentials (corresponding to ζ-potential between −10 and +10 mV), similar to those used in this work (apparent ζ-potential of −4 ± 1 mV in cell culture media), tend to enter the cell via lipid raft-mediated endocytosis. 55 While the Qdots used in this study caused no discernable decrease in cell viability (Fig.…”

“…55 It has been shown that quantum dots with lower surface potentials (corresponding to ζ-potential between −10 and +10 mV), similar to those used in this work (apparent ζ-potential of −4 ± 1 mV in cell culture media), tend to enter the cell via lipid raft-mediated endocytosis. 55 While the Qdots used in this study caused no discernable decrease in cell viability (Fig. S1 †), it has been shown that NPs can induce significant changes in cell function, such as changes in gene expression, even without detectable changes in cell viability.…”

“…The preferential co-localization with lipid rafts suggests that lipid raft-mediated endocytosis may play an important role in the uptake of these Qdots. The uptake of Qdots with similar ζ-potentials as those used in our study has been shown to occur via lipid raft-mediated endocytosis, 55 although the use of an inhibitor of this uptake mechanism was used in that study, as opposed to the direct, high resolution observation of co-localization that was applied in our study.…”

The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation

“…An upper size limit reported for particles entering via this pathway is 200 nm [8, 11]. Silica NPs of 200 nm and polymeric NPs of 100–200 nm were reported to internalize predominantly through CME [11, 28], and the positive charge on the surface of quantum dots [29], dendrimers [30] and polymer NPs [10, 31] was shown to increase the probability of internalization via CME rather than the use of other endocytic pathway. In our study, the inhibitors of CME were not very successful to inhibit the uptake of Rubipy-SiO 2 NPs by CaCo-2 cells; however the effect of chlorpromazine and the colocalisation with clathrin suggests a minor role of this pathway in the internalization of 30 nm Rubipy-SiO 2 NPs.…”

Synthetic Biodegradable Microparticle and Nanoparticle Vaccines against the Respiratory Syncytial Virus

“…In general, particles of 50–80 nm are endocytosed through caveolae-mediated endocytosis [43,44,45]; particles <150 nm in diameter are taken up via classic receptor-mediated endocytosis through clathrin-coated pits [45,46]; and larger particles (>500 nm) are endocytosed via macropinocytosis and phagocytosis and restricted to professional antigen presenting cells (APC), such as macrophages and immature DCs [38]. Particles can also be endocytosed via clathrin- and caveolae-independent lipid raft-mediated mechanisms, but the size-restrictions are not well understood [44,46]. Upon internalization, the particles are hydrolyzed in the endosome/lysosomes of APCs, and antigens are then processed and complexed with MHC class II molecules for subsequent presentation and activation of CD4 T cells [47,48].…”