Development of genistein-PEGylated silica hybrid nanomaterials with enhanced antioxidant and antiproliferative properties on HT29 human colon cancer cells

Abstract

The anticancer use of genistein (Gen) has been severely limited due to its low water solubility, low bioavailability, and instability under experimental conditions. To overcome these limitations, we propose a formulation of a hybrid nanomaterial (HNM) based upon the incorporation of Gen into PEGylated silica nanoparticles (PEG-SiNPs) (Gen-PEG-SiHNM), where their physicochemical and biological effects on HT29 cells were evaluated. Genistein-loaded PEGylated silica hybrid nanomaterials were obtained by a simple end effective aqueous dispersion method. Physicochemical properties were determined by its mean particle size, surface charge, amount of cargo, spectroscopic properties, release profiles and aqueous solubility. In vitro biological performance was carried out by evaluating its antioxidant capacity and elucidating its antiproliferative mechanistic. Results showed that small (ca. 33 nm) and spherical particles were obtained with positive surface charge (+9.54 mV). Infrared analyses determined that encapsulation of genistein was successfully achieved with an efficiency of 51%; it was observed that encapsulation process enhanced the aqueous dispersibility of genistein and cumulative release of genistein was pH-dependent. More important, after encapsulation data showed that Gen potentiated its antioxidant and antiproliferative effects on HT29 human colon cancer cells by the modulation of endogenous antioxidant enzymes and H₂O₂ production, which simultaneously activated two different processes of cell death (apoptosis and autophagy), unlike free genistein that only activated one (apoptosis) in a lower proportion. Overall, our data support that Gen-PEG-SiHNM could be potentially used as alternative treatment for colorectal cancer in a near future.

Keywords: Colorectal cancer; PEGylated silica nanoparticles; antiproliferative effects; genistein; hybrid nanomaterials; mechanisms of programmed cell death.

Figure 1

Chemical structure of genistein.

Figure 2

Schematic representation of the development of hybrid nanomaterials formed with genistein and PEGylated silica nanoparticles.

Figure 3

TEM micrographies of silica nanoparticles (A), PEGylated silica nanoparticles (B), and genistein-loaded PEGylated silica hybrid nanomaterials (C).

Figure 4

A. DRIFT spectra of APTES, PEG, genistein (Gen), silica nanoparticles (SiNPs), APTES-silica nanoparticles (APTES-SiNPs), PEGylated silica nanoparticles (PEG-SiNPs), and genistein-PEGylated silica hybrid nanomaterials (Gen-PEG-SiHNM). DRIFT spectrum represents the mean of 16 scans. B. Percentage of incorporated and not incorporated genistein into PEGylated silica nanoparticles (i), and cumulative release of genistein after 48 h of assessment in PBS:EtOH buffer at pH 7.2 and 6.5 (ii). All results represent the mean ± SE of five different batches examined by triplicate. Determinations were carried out by UV-Vis spectroscopy at 420 nm.

Figure 5

A. Spectrum UV-Vis of free and encapsulated genistein after filtration process through 0.45 µm syringe filters and dispersed in PBS 10 mM pH 7.2. B. Specific solubility of both free and encapsulated genistein in PBS 10 mM pH 7.2 as a function of temperature (37°C). Data shown the mean ± standard error of three different batches with at least five repetitions. Different letters indicate significant difference (P < 0.05) between samples.

Figure 6

Antioxidant capacity of silica nanoparticles (PEG-SiNPs), free genistein (Gen) and genistein-PEGylated silica hybrid nanomaterials (Gen-PEG-SiHNM) determined by (A) the oxygen radical absorbance capacity (ORAC) and (B) the Trolox equivalent antioxidant capacity (TEAC) assays. Results are expressed as mean ± SEM of three individual experiments by triplicate. Different letters indicate statistical differences (P < 0.05) among samples analyzed by one way ANOVA followed by Tukey test.

Figure 7

A. Cell viability (%) after 24 h of exposition to genistein (Gen), PEGylated silica nanoparticles (PEG-SiNPs), and genistein-PEGylated silica hybrid nanomaterials (Gen-PEG-SiHNM) determined by MTT assay. B. Lactate dehydrogenase (LDH) activity in HT29 cells after 24 h exposition with IC₅₀ of Gen, PEG-SiNPs and Gen-PEH-SiHNM. Each value represent the mean of three independent experiments by triplicate ± SE. *indicate significant difference in comparison with control by Dunnet’s test (P < 0.05).

Figure 8

TUNEL-positive cells observed after 24 h of exposition to genistein (Gen) and genistein-PEGylated silica hybrid nanomaterials (Gen-PEG-SiHNM) (AI). Results are expressed as the number of TUNEL-positive cells observed in at least 15 different fields (Mean ± SEM). Representative images of TUNEL staining detection of apoptotic cells observed in at least 15 different fields (200x magnification), where control (1 and 2), Gen (3 and 4), and Gen-PEG-SiHNM (5 and 6) were observed at bright (1, 3, and 5) and fluorescence (2, 4, and 6) fields (AII). Superoxide dismutase (SOD, BI) and Catalase (CAT,) activities found in HT29 cells after 24 h of exposition to IC₅₀ of Gen and Gen-PEG-SiHNM treatments (BII). Data represent the mean of at least two different experiments evaluated by triplicate ± SE. H₂O₂ production (µM) found in HT29 cells after 24 h of exposition to IC₅₀ of Gen and Gen-PEG-SiHNM treatments (C). The amount of PEG-SiNPs was the same used in Gen-PEG-SiHNM. *indicate statistical difference (P < 0.05) between treatments and control group analyzed by Dunnet’s test.

Figure 9

A. Autophagy’s activation in HT29 cells after 24 h of incubation with genistein (Gen) and Genistein-PEGylated silica hybrid nanomaterials (Gen-PEG-HNM) at IC₅₀ (24.3 μM). *indicate statistical difference (P < 0.05) between treatments and control group analyzed by Dunnet’s test. B. Autophagic structural analysis by TEM in HT29 cells exposed to 23.43 µM of free or encapsulated Gen for 24 h. Arrows show the presence of autolysosomes formed after cells were exposed to treatments for 24 h. Untreated HT29 cells were used as control.