Anticancer therapeutic potential of multimodal targeting agent- "phosphorylated galactosylated chitosan coated magnetic nanoparticles" against N-nitrosodiethylamine-induced hepatocellular carcinoma

Abstract

Superparamagnetic iron oxide nanoparticles (SPIONs) are extensively used as carriers in targeted drug delivery and has several advantages in the field of magnetic hyperthermia, chemodynamic therapy and magnet assisted radionuclide therapy. The characteristics of SPIONs can be tailored to deliver drugs into tumor via "passive targeting" and they can also be coated with tissue-specific agents to enhance tumor uptake via "active targeting". In our earlier studies, we developed HCC specific targeting agent- "phosphorylated galactosylated chitosan"(PGC) for targeting asialoglycoprotein receptors. Considering their encouraging results, in this study we developed a multifunctional targeting system- "phosphorylated galactosylated chitosan-coated magnetic nanoparticles"(PGCMNPs) for targeting HCC. PGCMNPs were synthesized by co-precipitation method and characterized by DLS, XRD, TEM, VSM, elemental analysis and FT-IR spectroscopy. PGCMNPs were evaluated for in vitro antioxidant properties, uptake in HepG2 cells, biodistribution, in vivo toxicity and were also evaluated for anticancer therapeutic potential against NDEA-induced HCC in mice model in terms of tumor status, electrical properties, antioxidant defense status and apoptosis. The characterization studies confirmed successful formation of PGCMNPs with superparamagnetic properties. The internalization studies demonstrated (99-100)% uptake of PGCMNPs in HepG2 cells. These results were also supported by biodistribution studies in which increased iron content (296%) was noted inside the hepatocytes. Further, PGCMNPs exhibited no in vivo toxicity. The anticancer therapeutic potential was evident from observation that PGCMNPs treatment decreased tumor bearing animals (41.6%) and significantly (p ≤ 0.05) lowered tumor multiplicity. Overall, this study indicated that PGCMNPs with improved properties are efficiently taken-up by hepatoma cells and has therapeutic potential against HCC. Further, this agent can be tagged with ³²P and hence can offer multimodal cancer treatment options via radiation ablation as well as magnetic hyperthermia.

Keywords: Asialoglycoprotein receptor; Liver cancer; NDEA; Superparamagnetic iron oxide nanoparticles; Targeted drug delivery.

Fig. 1

Size distribution, representative ζ -potential, high-resolution TEM images, X-ray diffraction patterns and vibrating sample magnetometry of PGCMNPs (A) I. Hydrodynamic size distribution of PGCMNPs (scale: 0–1000 nm), II. ζ-potential of PGCMNPs (Scale: -200–200 mV) (B) I. PGCMNPs were quasi-spherical in shape (scale: 50 nm) II. Mean particle size of PGCMNPs III. HR-TEM images of PGCMNPs with lattice fringes IV. SAED patterns of PGCMNPs (C) I. X-ray diffraction patterns of magnetite (Fe₃O₄) II. X-ray diffraction patterns of PGCMNPs. The peaks of PGCMNPs corresponding to the crystal planes of magnetite (Fe₃O₄) are marked as (°). The peak corresponding to PGC is marked as (*) (D) Magnetization curve for PGCMNPs displaying superparamagnetic properties. Remnant magnetization or remaining magnetization (M_r) and coercivity (H_c) was observed to be M_r≈0.41 emu/g and H_c≈63.3 Oe, respectively. The very negligible M_r and H_c values of PGCMNPs confirmed their superparamagnetic state

Fig. 2

FT-IR spectrum, surface morphology and elemental composition of PGCMNPs. A. FT-IR spectrum of PGCMNPs B. Scanning electron micrograph of PGCMNPs with spherical morphology C. The elements in PGCMNPs were identified to be carbon (24.67 ± 4.37%), nitrogen (1.33 ± 0.09%), oxygen (23.36 ± 1.88%), phosphorous (11.97 ± 1.97%) and iron (38.37 ± 4.37%) D. Probable structure of PGCMNPs

Fig. 3

In vitro antioxidant properties and hemolytic activity of PGCMNPs. A Ferric ion reducing power of PGCMNPs when compared with PGC B Ferrous ion chelating ability of PGCMNPs when compared with PGC C SOR scavenging activity of PGCMNPs when compared with PGC D Lipid peroxidation scavenging ability of PGCMNPs when compared with PGC. Results presented as mean ± SD (n = 6). ^$, ^€ and.^¥ signifies p ≤ 0.05 when compared with 0.25, 0.5, and 1 mg/mL of PGC, respectively E Hemolysis induced by PGCMNPs was < 5%, which was well within the acceptance criteria of ASTM E2524-08 standard. Results expressed as Mean ± SD (n = 3)

Fig. 4

H & E-stained sections of animals treated with PGCMNPs. I, II: Microphotographs of liver displaying normal arrangement of hepatocytes (HP) with no condensation and regeneration of cytoplasm. Central vein (CV) and sinusoids (SN) were normal in appearance and RBC pooling was not observed. III, IV: Microphotographs of spleen were normal with distinguishable white pulp (WP), red pulp (RP) and marginal zones (MZ). V,VI: Microphotographs of testis displaying normal appearance of seminiferous tubules (ST) with sperm (S), spermatogonia cells (SG) bordered with peritubular myoid cells (MC) and groups of Leydig cells (LC). VII,VIII: Microphotographs of kidney with regular architecture displaying glomerulus (G), proximally convoluted tubule (PCT) and distally convoluted tubule (DCT)

Fig. 5

Flow cytometry images of PGCMNPs uptake: (A) Gating strategies for the experiment (B) Control HepG2 cells without PGCMNPs treatment (blue color is represented as HepG2 cells with no fluorescence). Representative results of HepG2 cells treated with (C) FITC-Chitosan (D) FITC-Phosphorylated Chitosan (E) FITC-PGC (F) FITC-PGCMNPs for 24 h (Blue color is presented as cells with no fluorescence; purple color is presented as cells with fluorescence)

Fig. 6

Biodistribution of PGCMNPs in the liver and blood serum. A Representative photomicrographs of liver stained with Prussian blue to detect iron deposits at 1 h, 3 h and 24 h after the intravenous administration of PGCMNPs (400X) B Changes in the Fe levels of liver at 1 h, 3 h and 24 h after the intravenous injection of PGCMNPs C Changes in the Fe levels of blood serum at 1 h, 3 h and 24 h after the intravenous injection of PGCMNPs. Data were presented as Mean ± SD

Fig. 7

In vivo anticancer therapeutic potential of PGCMNPs: (A) Liver morphology after 4 weeks of PGCMNPs therapy. (I, II) Control and PGCMNPs groups displaying normal and distinct lobes (III) Liver in the TUMOR group displaying enlarged lobes with HCC nodules ≥ 3 mm (arrowhead) (IV) TUMOR + PGCMNPs group displaying abnormal liver with small tumors (arrowhead) (B) H & E-stained sections of liver (I, II) Histopathology of normal Control and PGCMNPs groups displaying normal arrangement of hepatocytes (III) Histopathology of TUMOR group displaying moderately differentiated HCC (IV) Histopathology of TUMOR + PGCMNPs group displaying well-differentiated tumor (magnification 100X). (C & D) TUNEL assay in liver/liver tumors. (C) (I and II) Photomicrographs of Control and PGCMNPs groups showing green stained nucleus in non-apoptotic cells (Arow) (III) Photomicrographs of TUMOR group showing brown-colored stains in nucleus and cytoplasm representing necrotic cells (arrow head) (IV) Photomicrograph of TUMOR + PGCMNPs group showing brown colored nucleus in cells undergoing apoptosis (circle) (magnification: 100X, scale bar: 50 µM) (D) Percentage of apoptotic cells in liver/liver tumors after 4 weeks of PGCMNPs treatment in TUMOR group animals (Data were expressed as mean ± SD and analysed using one-way ANOVA followed by post hoc test (Tukey’s test). *: represents (p ≤ 0.05) when compared with the Control group; ϯ: represents (p ≤ 0.05) when compared with PGCMNPs group; #: represents (p ≤ 0.05) when compared with TUMOR group)

Fig. 8

Mechanism of action of PGCMNPs. PGCMNPs were internalized through clathrin mediated endocytosis and further degraded into free ferrous and ferric ion in the lysosomes. The released iron gets accumulated in the cytosol and mitochondria and increase oxidative stress through Fenten and Haber- Weiss reaction. The oxidative stress further leads to peroxidation of polyunsaturated lipids in membrane and modifications of the transporters on the transporters on the membrane. The decreased uptake of cysteine for the formation of glutathione and increased lipid peroxidation leads to higher oxidative stress in mitochondria and ferroptosis