Two-Step Preparation of Protein-Decorated Biohybrid Quantum Dot Nanoparticles for Cellular Uptake

Abstract

Decoration of nanoparticles with specific molecules such as antibodies, peptides, and proteins that preserve their biological properties is essential for the recognition and internalization of their specific target cells. Inefficient preparation of such decorated nanoparticles leads to nonspecific interactions diverting them from their desired target. We report a simple two-step procedure for the preparation of biohybrid nanoparticles containing a core of hydrophobic quantum dots coated with a multilayer of human serum albumin. These nanoparticles were prepared by ultra-sonication, crosslinked using glutaraldehyde, and decorated with proteins such as human serum albumin or human transferrin in their native conformations. These nanoparticles were homogeneous in size (20-30 nm), retained the fluorescent properties of quantum dots, and did not show a "corona effect" in the presence of serum. The uptake of transferrin-decorated quantum dot nanoparticles was observed in A549 lung cancer and SH-SY5Y neuroblastoma cells but not in non-cancerous 16HB14o- or retinoic acid dopaminergic neurons differentiated SH-SY5Y cells. Furthermore, digitoxin-loaded transferrin-decorated nanoparticles decreased the number of A549 cells without effect on 16HB14o-. Finally, we analyzed the in vivo uptake of these biohybrids by murine retinal cells, demonstrating their capacity to selectively target and deliver into specific cell types with excellent traceability.

Keywords: albumin; drug delivery; endocytosis; nanoparticles; transferrin.

Figure 1

Preparation of decorated NPs. The first step is the preparation of a biphasic system, followed by the application of ultrasound, and phase separation by centrifugation. The process takes 20 min and provides NPs in an aqueous phase, with their surface activated. In the second step, NPs were decorated by the addition of a protein or peptide of interest to the solution and incubating ON at 4 °C followed by centrifugation at 10,000 rpm. The picture shows the resuspended, water-soluble decorated HSA-QDs illuminated with UV light, with turquoise (450 nm), green (525 nm), yellow (570 nm), and hTf-QDs with red (630 nm), and infrared (780 nm) emissions.

Figure 2

Characterization of decorated NPs. The dynamic light scattering (DLS) plots show the relative mass and media diameter of HSA-QDs (A) and hTf-QDs (D). The insets are TEM images (100,000×) of HSA-QDs (B) and hTf-QDs (E), respectively, after uranyl acetate staining. The scheme in (C) represents the structure of the NPs obtained with this procedure. (F) Absorbance (RFU = Relative Fluorescence Units) spectra of HSA-QDs (green) and hTf-QDs (red) obtained using a Nanodrop 1000 spectrophotometer.

Figure 3

The external monolayer shell of decorated NPs preserves protein function. (A) Left panel: UV-illuminated agarose gel electrophoresis of HSA-QDs (lane 2) and hTf-QDs (Lane 3). HSA-NPs without QDs were used as control (lane 1). Right panel: UV-illuminated agarose gel electrophoresis of HSA-QDs (lane 4) and heat denatured (5 min at 95 °C) ones (HSAQDsDnt, lane 5). Free amines were labeled with FSC to visualize HSA (green), hTf (green). QDs are observed by their own emission (red). Lane 6 shows a DNA ladder (100–3000 bps) visualized by the presence of ethidium bromide (red). (B). Activity (brown color) of AP-QDs (Tube 3, AP-QDs). Reactions without enzyme (yellow color, tube 1) or with AP (1 mg/mL, tube 2) were used as negative and positive control, respectively. (C) Average diameter of hTf-QDs in PBS alone or PBS + FBS 50% demonstrating lack of corona effect.

Figure 4

Biohybrid decorated NPs preserve the fluorescent properties of QDs (A) and can be quantitatively measured by fluorimeter (B), fluorescence microscopy (C), or image analysis (D). (A) Left panel: Fluorescence (630 nm emission) of 20-mL flasks containing HSA-NPs (left) and HSA-QDs (right) illuminated with UV light at 350 nm. Right panel: absorbance spectra plot of HSA-QDs measured using a Nanodrop 3300 fluorimeter. (B) Fluorescent quantification (Relative Fluorescent Unit, RFU) of HSA-QDs excited with white light, UV (280–350 nm) light, and blue (490 nm) light measured on Nanodrop 3300. (C) Representative microscope fluorescent images of diluted solutions (10⁻² and 10⁻⁴) dried onto coverslips of HSA-QDs. (D) Quantification of HSA-QDs samples by image analysis. A serial dilution (10⁻¹–10⁻⁸) of QDs was used as standard. UV excited fluorescence of QDs was captured by a LAS 500 digital analyzer and quantified by extrapolation using Image-J software.

Figure 5

Early and selective uptake of HSA-QDs and hTf-QDs in neuroblastoma cells. Human SH-SY5Y neuroblastoma line differentiated into dopaminergic neurons by retinoic acid (+RA) does not internalize HSA-QDs (A) or hTf-QDs (B) after 72 h incubation. In contrast, SH-SY5Y neuroblastoma cells show strong fluorescent signals after only 3 h of incubation with HSA-QDs (C) or hTf-QD (D). (E,F): UV Fluorescence signals of (C,D), without contrast light. Red arrows indicate nonspecific NP interactions. Bars = 50 µm.

Figure 6

A549 lung cancer cells internalize HSA-QDs and hTf-QDs. Fluorescence microscopy representative images of A549 cells. Fluorescence emitted with ultraviolet light (UV) (First column; (A,D,G)), phase contrast (second column; (B,E,H)) and merge (third column; (C,F,I)). The first row shows cells incubated without NPs. The second and third rows were incubated with HSA-QDs and hTf-QDs, respectively. Scale bars = 20 μm.

Figure 7

Energy-dependent internalization of HSA-QDs. Reconstruction of 3D images captured by confocal microscopy after incubation for 3 h with HSA-QDs at 4 °C (A,C) or 37 °C (B,D), respectively. HSA-QDs and DAPI-stained nuclei are shown as red and blue, respectively. Scale bars = 20 μm.

Figure 8

Temperature-dependent incorporation of hTf-QDs is higher than HSA-QDs in A549 cells. Cells were co-incubated with hTf-QDs (red) and HSA-QDs (green) for 3 h. (A) Representative plot showing the frequency of the number of NPs per A549 cell (quantification of three independent experiments are shown in panels (E,F)). (B–D) Representative fluorescence microscopy images using UV and transmission light simultaneously of A549 cells at 4 °C (B), 37 °C (C), and 16HB14o- cells at 37 °C (D). The co-localization of two HSA-QDs and hTf-QDs is visualized in yellow (arrow). DAPI-stained nuclei are shown in blue. (E) Box plot of HSA-QDs and hTf-QDs per A549 cell incubated at 4 °C or 37 °C during 3 h. The box comprises the 2nd and 3rd quartile of the data and the horizontal thick line defines the median. Outliers (>1.5× interquartile range are marked by diamonds). (F) Box plot of the average number of HSA-QDs and hTf-QD per 16HB14o- cell incubated at 37 °C for 3 h.

Figure 9

Digitoxin-loaded hTf-QDs reduced A549 cell number. Left panel: Representative fluorescence microscopy images (20×) of 16HB14o- and A549 cells incubated for 48 h without (Control, (A,B) panels) or with hTf-QDs ((C,D) panels). hTf-QDs are visualized in red and DAPI-stained nuclei are shown in blue. (E) Effect of hTf-QDs loaded with digitoxin (hTf-QDs-Dig) on 16HB14o- (green) and A549 (red) cells treated for 48 h. As a control, cells were incubated without hTf-QDs (Control) or in the presence of hTf-QDs without digitoxin (hTf-QDs). * indicates significative difference between treatments.

Figure 10

Uptake of HSA-QDs (Panel (A)) and hTf-Qs (panel (B)) by ARPE-19 cells. The 3D reconstruction demonstrates HSA-QDs aggregates in the cytoplasm (Panel (C)). All images (merge) were taken with red and blue (DAPI) filters. Scale bar = 20 μm.

Figure 11

In vivo circulation of decorated HSA-QDs and hTf-Qs by RPE cells via subconjunctival injection. (A). Schematic of the eye anatomy and retina and subconjunctival injection of NPs. GCL = Ganglion Cell Layer, INL = Inner Nuclear Layer, ONL = Outer Nuclear Layer, RPE = retinal pigment epithelium (B). Control tissue of eye inoculated via subconjunctival injection with PBS showing spontaneous autofluorescence (BV = Blood vessel). (C,D) Tissue inoculated via subconjunctival injection with HSA-QDs and hTf-QDs, respectively. All images were taken with a red filter and phase contrast (merge). Scale bar = 50 μm.

Figure 12

In vivo cellular uptake by RPE cells of HSA-QDs (green filter, Panel (A)) and hTf-QDs (red filter, Panel (B)) inoculated via subconjunctival injection. Panel (C) shows the merge image of HSA-QDs, hTf-QDs, and DAPI-stained nuclei (blue filter). Panel (D) shows the merge image of DAPI + Phase contrast. Panel (E) is a magnification of the inset shown in (C). White arrowheads show DAPI-stained nuclei. White arrows show intracellular co-localization of HSA-QDs and hTf-QDs (yellow dots).