Crossing the blood-brain barrier with carbon dots: uptake mechanism and in vivo cargo delivery

Abstract

The blood-brain barrier (BBB) is a major obstacle for drug delivery to the central nervous system (CNS) such that most therapeutics lack efficacy against brain tumors or neurological disorders due to their inability to cross the BBB. Therefore, developing new drug delivery platforms to facilitate drug transport to the CNS and understanding their mechanism of transport are crucial for the efficacy of therapeutics. Here, we report (i) carbon dots prepared from glucose and conjugated to fluorescein (GluCD-F) cross the BBB in zebrafish and rats without the need of an additional targeting ligand and (ii) uptake mechanism of GluCDs is glucose transporter-dependent in budding yeast. Glucose transporter-negative strain of yeast showed undetectable GluCD accumulation unlike the glucose transporter-positive yeast, suggesting glucose-transporter-dependent GluCD uptake. We tested GluCDs' ability to cross the BBB using both zebrafish and rat models. Following the injection to the heart, wild-type zebrafish showed GluCD-F accumulation in the central canal consistent with the transport of GluCD-F across the BBB. In rats, following intravenous administration, GluCD-F was observed in the CNS. GluCD-F was localized in the gray matter (e.g. ventral horn, dorsal horn, and middle grey) of the cervical spinal cord consistent with neuronal accumulation. Therefore, neuron targeting GluCDs hold tremendous potential as a drug delivery platform in neurodegenerative disease, traumatic injury, and malignancies of the CNS.

Fig. 1. Flowchart of the methods for in vitro and in vivo studies. GluCDs were used for in vitro studies and GluCD-F was used for in vivo studies.

Fig. 2. (a) Schematic representation of the bottom-up carbon dot synthesis via hydrothermal carbonization of d-glucose in a TEFLON-lined autoclave reactor at 200 °C for 5 h (GluCDs). (b) Schematic representation of the conjugation of 5-amionomethyl-fluorescein to glucose carbon dots (GluCD-F) using EDC/NHS coupling reaction. Structures of EDC, NHS and 5-aminomethyl-fluorescein is given on the bottom of the figure.

Fig. 3. Characterization of GluCDs and GluCD-F. (a) UV/Vis spectra of GluCDs and GluCD-F, (b) PL emission spectrum of GluCDs show excitation dependent PL emission. The maximum emission peak was at 450 nm when excited at 350 nm. Inset shows the normalized PL emission. (c) PL emission spectrum of GluCD-F. The PL emission of the conjugate is excitation independent. The maximum emission peak was at 526 nm when excited at 500 nm. (d) FTIR-ATR spectra of d-glucose (purple), GluCDs (black) and GluCD-F conjugate (red). (e) TEM image of GluCDs. Scale bar represents 10 nm. (f) Particle size distribution histogram of GluCDs.

Fig. 4. (a) Growth curves of all groups in three replicates: black curves are EBY.VW 5000-C, red curves are EBY.VW 5000-T, green curves are BC-C and orange curves are BC-T (b) bar graph shows the mean growth rates (OD₆₆₀/h) of EBY.VW 5000-C, EBY.VW 5000-T, BC-C and BC-T with standard error of the mean. Growth rates of treatment groups show no significant difference compared to respective controls (p > 0.05), (c) representative confocal images of EBY.VW 5000 and BC strains' control and treatment groups under bright field and fluorescence channels, merged images as well as enlarged images. Only BC-T shows bright fluorescence when treated with GluCDs, (d) bar graph shows the mean fluorescence intensity of all groups based on the quantitative fluorescence intensity analysis using confocal images. The table shows p-value for comparisons between each group. Fluorescence intensity of the treatment group of BC (Hex+) strain (BC-T) is significantly higher than that of all other groups (*p < 0.05) and there is no significant difference in the fluorescence intensity between other groups. (e) Delta fluorescence intensity of treatment groups of BC-T-C (Hex+) and EBY.VW 5000-T-C (Hex−) strains after subtracting the corresponding autofluorescence, ***p value < 0.0005.

Fig. 5. (a) Confocal images of wild-type zebrafish showing the injection route, heart, blood stream, CNS and observation area (central canal of spinal cord). (b) Accumulation of GluCDs-F in the CNS of zebrafish. The yellow arrow indicates the central canal of spinal cord of zebrafish.

Fig. 6. GluCD-F was observed in different regions of rat CNS. Sample images show GluCD-F localization in the ventral horn of the cervical spinal cord (top), middle grey matter of the cervical spinal cord (middle), and dorsal horn of cervical spinal cord (bottom). Corresponding cervical spinal cord cross-section camera obscura schematic showing the area of the image taken are displayed at the left column. Scale bar: 50 μm.