Development of asialoglycoprotein receptor directed nanoparticles for selective delivery of curcumin derivative to hepatocellular carcinoma

Abstract

Hepatocellular cellular carcinoma (HCC) is one of the most challenging liver cancer subtypes. Due to lack of cell surface biomarkers and highly metastatic nature, early detection and targeted therapy of HCC is an unmet need. Galactosamine (Gal) is among the few selective ligands used for targeting HCCs due to its high binding affinity to asialoglycoprotein receptors (ASGPRs) overexpressed in HCC. In the present work, we engineered nanoscale G4 polyamidoamine (PAMAM) dendrimers anchored to galactosamine and loaded with the potent anticancer curcumin derivative (CDF) as a platform for targeted drug delivery to HCC. In vivo targeting ability and bio-distribution of PAMAM-Gal were assessed via its labeling with the clinically used, highly contrast, near infrared (NIR) dye: S0456, with testing of the obtained conjugate in aggressive HCC xenograft model. Our results highlighted the targeted dendrimer PAMAM-Gal ability to achieve selective high cellular uptake via ASGPR mediated endocytosis and significantly enhance the delivery of CDF into the studied HCC cell lines. Cytotoxicity MTT assays in HCC cell lines, interestingly highlighted, the comparative high potency of CDF, where CDF was more potent as a chemotherapeutic anticancer small molecule than the currently in use Doxorubicin, Sorafenib and Cisplatin chemotherapeutic agents. In conclusion the proof-of-concept study using nanoscale PAMAM-Gal dendrimer has demonstrated its competency as an efficient delivery system for selective delivery of potent CDF for HCC anticancer therapy as well as HCC diagnosis via NIR imaging.

Keywords: Materials science; Pharmaceutical chemistry; Pharmaceutical science.

Fig. 1

PAMAM-Succ Synthesis. Opening of the anhydride ring in succinic anhydride acids (1) by terminal primary amine groups on the surface of G4 PAMAM dendrimers (2) to form amide bond links and obtain the PAMAM-Succ product (3). The reaction mixture was stirred in DMSO at 55 °C for 24 hours.

Fig. 2

PAMAM-Gal Synthesis. Coupling of sulfo N-hydroxysuccinimide esters of PAMAM-Succ with amine group in galactosamine. HCl (4) in its basic form. The reaction was performed at pH 7.4 for 24 hours. Amide bonds were formed and PAMAM-Gal product (5) was obtained.

Fig. 3

Overlay of the FT/IR spectra of PAMAM-Succ (green), PAMAM-Gal (blue), Galactosamine (red), and G4 PAMAM (greenish yellow). Characteristic peaks are marked as shown on the spectra.

Fig. 4

Proton nuclear magnetic resonance (¹H NMR) spectra of: (A) G4 PAMAM (B) PAMAM-Succ (C) GAL (D) PAMAM-GAL. The ¹H NMR spectrum of PAMAM-GAL showed the characteristic integrated peaks as described. Characteristic peaks of PAMAM dendrimers and their products are included.

Fig. 5

(A & B) Hydrodynamic size of plain and targeted dendrimers in nanometers by (DLS); (C & D) Morphology and size measurements of G4 PAMAM and PAMAM-GAL dendrimers characterized by TEM are shown, with scale bar = 20 nm, and 100 nm respectively; and (E & F) Zeta potential measurements of both dendrimers in millivolts.

Fig. 6

(A) Cytotoxicity assay of Plain Drugs after 48h (B) Cytotoxicity Assay of free CDF, G4-PAMAM-CDF, and G4-PAMAM-GAL-CDF after 48h (C) Cytotoxicity Assay of plain PAMAM, PAMAM-GAL showed that they were safe at higher concentrations and conjugation with GAL reduced the cytotoxicity. Data represented as mean (±SD, n = 6). (D) Summary of IC50s is represented using GraphPad Prism software® as shown. The histogram columns represent means of three independent experiments with 6 replicates for each treatment; bars, S.E. Figures (A–C), p ≤ 0.05* and ** statistically significant inhibition (*p ≤ 0.05) relative to DMSO-treated respective controls or (**p ≤ 0.05) relative to CDF drug treated cells.

Fig. 7

Fluorescence microscopic study. Cancer cell selective uptake of Rhodamine B labeled targeted dendrimers than the non-targeted dendrimers in HepG2 cells after 2 h treatment. Blue and red fluorescence indicate cell nuclei and Rhodamine B, respectively.

Fig. 8

HCC imaging of targeted formulation. (A) Whole body NIR fluorescence imaging of targeted PAMAM-Gal-S0456. Ex-vivo imaging of tumor (B), liver (C), kidney (D) after 14 h of post i.v injection, and (E) control-nanoparticle-dye treated mice. Higher tumor uptake was found in PAMAM-Gal-S0456 as compared to control dye treated mouse. The higher intensity of kidney as compared to liver indicates that PAMAM-Gal-S0456 is been excreted out through the kidneys.