Letter pubs.acs.org/JPCL
Clathrin to Lipid Raft-Endocytosis via Controlled Surface Chemistry and Efficient Perinuclear Targeting of Nanoparticle Atanu Chakraborty and Nikhil R. Jana* Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata 700032, India S Supporting Information *
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 raftmediated 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 raftmediated 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.
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subcellular trafficking.34,35 This interaction depends on the nanoprobe surface charge,36 lipophilicity, 37,38 size,39−42 shape,43−45 and cell type.46,47 There are significant computation studies on cell−nanoparticle interaction with the particular focus on the effect of nanoparticle size, shape, and surface chemistry.44,48 It is shown that endocytosis of nanoparticle follows by wrapping processes with cell membrane that involves membrane-particle adhesion, elastic deformation of membrane, and receptor diffusion on the membrane surface.48 All of these processes lead to size, shape, and surface chemistry effects of the nanoparticle. Hydrophobic nanoparticles can be inserted on the membrane as they prefer lipid tails but cannot cross the membrane. In contrast, charged nanoparticles may cross the membrane by pore formation after wrapping with the membrane. The receptor-mediated endocytosis of nanoparticle involves stronger specific interaction with membrane that depends on the strength of ligand receptor interaction and ligand density. Experimental work showed that the cellular internalization process of nanoprobes is highly sensitive to their surface chemistry. For example, cationic nanoparticles are readily internalized into cells as compared to anionic nanoparticles;36
anoparticle-based bioimaging probes are a promising alternative of conventionally used molecular probes.1−4 These nanoprobes are optically bright, stable, and with tunable colors from visible to near-infrared range. A variety of imaging nanoprobes have been developed which are made of quantum dots,1 doped semiconductor nanoparticles,5 iron oxide nanoparticles,3 gold cluster/nanoparticles,2,6 silicon nanoparticles,7,8 and fluorescent carbon nanoparticles.9 They have been used for the detection of cell surface receptors,1 extracellular molecules,1 cellular events,1 and as delivery carriers.10 These nanoprobes show potential for studying the cellular activities at the subcellular length scale, down to a single molecule label.11,12 However, the cellular interaction of these nanoprobes is significantly different as compared to molecular probes.4,13,14 This is due to their larger size and multivalent nature of interaction with cells.13,14 As a result, the cellular entry of nanoprobe occurs via endocytosis.15−33 Most works show that nanoprobe entry occurs via predominate clathrin-mediated endocytosis15−21 and in certain selected cases via predominate caveolae-mediated endocytosis22−30 or via macropinocytosis. 31,32 Some studies show that multiple endocytosis mechanisms are operative for a single nanoparticle.17,18,20,22,30,33 Subcellular imaging studies show that most of these nanoprobes are predominantly trafficked to endozomes/lysozomes.15−33 The interaction of nanoprobes with the lipidic cell membrane plays a critical role in the endocytosis processes and subsequent © 2015 American Chemical Society
Received: August 10, 2015 Accepted: September 3, 2015 Published: September 3, 2015 3688
DOI: 10.1021/acs.jpclett.5b01739 J. Phys. Chem. Lett. 2015, 6, 3688−3697
Letter
The Journal of Physical Chemistry Letters Scheme 1. Synthetic Approach for the Preparation of Four Different QDs
reported triphenylphosphonium functionalized quantum dot nanoprobes as mitochondria imaging probes and observed that they enter into cell predominantly via caveolae-mediated endocytosis which offers endosomal escape and subcellular targeting.53 This result prompted us for fine-tuning of nanoprobe surface chemistry and to investigate their role in a cell uptake mechanism. In this work, we have tuned the nanoprobe surface chemistry with the intention that they interact differently with cell membrane and would induce different cell uptake mechanism. We found that mechanism of cellular entry of nanoprobes can be shifted from predominate clathrin- to predominate lipid raft-mediated endocytosis by varying the surface chemistry and their lysozomal trafficking can be minimized. In particular, we have synthesized polyacrylate coated quantum dot (QD) with different chemical functional groups that produces functional QD with varying charge and lipophilicity. We found that cationic and zwitterionic QD enter into cell following predominate clathrin and partial lipid raft endocytosis. As lipophilic groups are introduced on weakly charged zwitterionic QD surface, their cell entry mechanism shifts to predominate lipid raft endocytosis. This shifting of endocytosis mechanism minimizes the lysosomal trafficking of QD and transports them to perinuclear regions, particularly to Golgi apparatus. This concept of surface chemistry dependent nanoparticle uptake can be extended for the development of different subcellular nanoprobes. Details of synthesis are shown in Scheme 1. First, a ZnS capped CdSe-based red emissive quantum dot (QD) has been synthesized via high-temperature organometallic approach, and then hydrophobic QD is converted to hydrophilic QD by a polyacrylate coating strategy reported elsewhere.52,53 Four
the lipophilic functional group enhances the interaction of nanoparticles with cells;37 polyethylene glycol functionalization minimizes nonspecific interaction with cells;49 and polyethylenimine type functionalization enhances subcellular targeting performance.50 These studies show two general strategies for subcellular targeting. In the first approach, the surface chemistry of nanoprobe is appropriately designed so that it can offer endosomal escape of the nanoprobe.34 This is particularly important as most of the nanoprobes enter into cells via predominate clathrin-mediated endocytosis and are trafficked to lysozomes. In the second approach, the nanoprobe is appropriately designed so that it can enter into cells via nonclathrin-mediated endocytosis, bypass endosomal trafficking, and target different subcellular organelles. For example, amphiphilic nanoparticle are designed for preferential lipid raft/ caveolae- mediated endocytosis,24−26,28,29 and
