Stimuli-responsive multifunctional glyconanoparticle platforms for targeted drug delivery and cancer cell imaging

Abstract

Targeted bioimaging or chemotherapeutic drug delivery to achieve the desired therapeutic effects while minimizing side effects has attracted considerable research attention and remains a clinical challenge. Presented herein is a multi-component delivery system based on carbohydrate-functionalized gold nanoparticles conjugated with a fluorophore or prodrug. The system leverages active targeting based on carbohydrate-lectin interactions and release of the payload by biological thiols. Cell-type specific delivery of the activatable fluorophore was examined by confocal imaging on HepG2 cells, and displays distinct selectivity towards HepG2 cells over HeLa and NIH3T3 cells. The system was further developed into a drug delivery vehicle with camptothecin (CPT) as a model drug. It was demonstrated that the complex exhibits similar cytotoxicity to that of free CPT towards HepG2 cells, and is significantly less cytotoxic to normal HDF and NIH3T3 cells, indicating excellent specificity. The delivery vehicle itself exhibits excellent biocompatibility and offers an attractive strategy for cell-type specific delivery depending on the carbohydrates conjugated in the system.

Scheme 1. Chemical structures of the activatable fluorophore NA-S-BA and the prodrug CPT-S-BA and the schematic diagram of their conjugation with gold nanoparticles.

Fig. 1. Absorption and emission changes of (A, B) NA-S-BA (10 µM) and (C, D) control NA-C-BA (10 µM) in the presence of GSH in DMSO/PBS solution (1 : 1, v/v, pH = 7.4, 10 mM). Insets (A) and (C): color changes observed in NA-S-BA and NA-C-BA solutions upon addition of GSH. Insets (B) and (D): visible fluorescence changes in NA-S-BA and NA-C-BA upon addition of GSH. Each point was recorded after exposure to GSH for 1 h at 37 °C, λ _ex = 405 nm. Note: here the isosbestic point of 405 nm is chosen as the excitation wavelength for an accurate comparison of the fluorescence intensity before and after GSH-induced cleavage of the disulfide bond.

Fig. 2. (A) Changes in fluorescence intensity at 535 nm for NA-S-BA and NA-C-BA (10 µM) in DMSO/PBS solution (1 : 1, v/v, pH = 7.4, 10 mM) in the presence (black and blue) and absence (red and purple) of GSH (500 eq.) over time, λ _ex = 405 nm. Data was recorded every 0.5 s. (B) Fluorescence response of NA-S-BA (10 µM) upon addition of various amino acids including Ala, Leu, Ile, Val, Pro, Phe, Met, Trp, Gly, Ser, Gln, Thr, Asn, Tyr, Asp, Glu, Lys, Arg, and His (500 eq.). Each spectrum was recorded after exposure to GSH for 1 h at 37 °C, λ _ex = 405 nm.

Scheme 2. Proposed fluorescence and CPT release mechanism by treatment with GSH.

Fig. 3. Concentration-dependent uptake of Au-Gal-BA in HepG2 cells as determined by flow cytometry. (A) Histograms of HepG2 cells with different concentrations of Au-Gal-BA. (B) Relative fluorescence intensities expressed with respect to control cells as mean ± SD (n = 3). Measured using flow cytometry (AmCyan channel, BD 525/50 filter).

Fig. 4. Confocal microscopy images of (A) HepG2, (B) HeLa and (C) NIH3T3 cells incubated with Au-Gal-BA. Cells were treated with the complexes for 2 h and the cytoskeletons were stained with Alexa Fluor 633 phalloidin. Cell images were acquired using excitation wavelengths of 488 nm and 633 nm, and emission filters in the ranges of 501–602 nm and 638–747 nm for the imaging of Au-Gal-BA (green) and phalloidin (red), respectively. The last panel shows the overlay of both channels.

Fig. 5. (A) Comparative viability of HepG2, NIH3T3 and HDF cells incubated with increasing concentrations of the Au-Gal-BA(CPT) complex for 72 h. Comparison of cell viability in (B) HepG2, (C) NIH3T3 and (D) HDF cells with increasing concentrations of CPT, CPT-S-BA, Au-Gal and Au-Gal-BA(CPT). Measured using WST-1 assay, with absorbance quantified at 450 nm (reference: 650 nm). Data is represented as mean ± SEM (n ≥ 3).

Fig. 6. Annexin V/PI assay of HepG2 control, HepG2 incubated with Au-Gal-BA(CPT), and CPT. Fluorescence was analyzed via flow cytometry (PE-CF594 and FITC channel). Inserted numbers indicate percentage of cells in each area.