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Elucidating the mechanism of cellular uptake and removal of protein

Overall, this work demonstrates a facile method to cluster nanoparticles without chemical modification and the possibility of developing nanocarriers of various sizes that can be used as nanobiomedicine.1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and L-α-phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) were obtained from Avanti Polar Lipids, Inc. A solution of 20 nm gold nanoparticles (6 × 10 particles per milliliter, stabilized suspension in citrate buffer) was purchased from Sigma-Aldrich (Steinheim, Germany).

All solutions were prepared using ultrapure water, obtained using a Millipore Milli-Q water purification system (Darmstadt, Germany).

Modulation of the lipid membrane curvatures influences not only the stability of the oligomeric state of the Au CLs, but also the rate of cellular uptake.

Dynamic light scattering (DLS) data showed that 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), with its relatively small head group, is crucial for establishing an effective membrane curvature to encapsulate the Au CLs.

Transmission electron microscopy (TEM) (JEM ATM 200F, JEOL, 200 k V) and UV-vis spectrometer was used to characterize the formation of Au CLs.

The size distribution of Au CLs, according to time, was measured by dynamic light scattering (DLS) (ZEN3690, Nano-ZS90, Malvern, UK).

Resin was removed by filtration, and the remaining solution was vaporized under nitrogen gas.

Furthermore, when cells were treated with Au CLs, hundreds of particles found inside the cells proved the ability of cellular uptake.

After lyophilizing for 2 days, distilled water was slowly added until it reached the desired volume, and the solution was sonicated in order to produce homogeneous uniformly sized small unilamellar vesicles (SUV). The solution was centrifuged at 9300 rcf for 10 min, and the supernatant, kept in ice, was used for further experiments.

Phospholipid-coated gold nanoparticles were prepared by mixing a solution of gold nanoparticles and SUV in a volume ratio of 1 : 9 and incubating the mixture at 4°C for 12 hours.

In order to thoroughly mix the two components, an inverting motion was applied with a rotating mixer.

To isolate Au CLs from the mixture of single gold nanoparticles and SUV, the sample solution was centrifuged at 12,000 rpm for 10 min, and the supernatant was removed.

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