Utilization of Enzyme-Immobilized Mesoporous Silica Nanocontainers (IBN-4) in Prodrug-Activated Cancer Theranostics

Abstract

To develop a carrier for use in enzyme prodrug therapy, Horseradish peroxidase (HRP) was immobilized onto mesoporous silica nanoparticles (IBN-4: Institute of Bioengineering and Nanotechnology), where the nanoparticle surfaces were functionalized with 3-aminopropyltrimethoxysilane and further conjugated with glutaraldehyde. Consequently, the enzymes could be stabilized in nanochannels through the formation of covalent imine bonds. This strategy was used to protect HRP from immune exclusion, degradation and denaturation under biological conditions. Furthermore, immobilization of HRP in the nanochannels of IBN-4 nanomaterials exhibited good functional stability upon repetitive use and long-term storage (60 days) at 4 °C. The generation of functionalized and HRP-immobilized nanomaterials was further verified using various characterization techniques. The possibility of using HRP-encapsulated IBN-4 materials in prodrug cancer therapy was also demonstrated by evaluating their ability to convert a prodrug (indole-3- acetic acid (IAA)) into cytotoxic radicals, which triggered tumor cell apoptosis in human colon carcinoma (HT-29 cell line) cells. A lactate dehydrogenase (LDH) assay revealed that cells could be exposed to the IBN-4 nanocomposites without damaging their membranes, confirming apoptotic cell death. In summary, we demonstrated the potential of utilizing large porous mesoporous silica nanomaterials (IBN-4) as enzyme carriers for prodrug therapy.

Keywords: Institute of Bioengineering and Nanotechnology (IBN-4); anti-cancer; enzyme immobilization; mesoporous silica nanoparticles; prodrug therapy.

Figure 1

Schematic representation of enzyme prodrug therapy using IBN-4-HRP nanocomposites in the presence of indole-3-acetic acid and the resultant cell apoptosis (1. Skatolyl radical and 2. Peroxyl radical).

Figure 2

Transmission electron microscopic images of IBN-4 nanoparticles: (a) As-synthesized IBN-4, (b) IBN-4-extracted, (c) IBN-4-NH₂, and (d) IBN-4-HRP.

Figure 3

(A) Nitrogen adsorption–desorption isotherms of (a) as-synthesized IBN-4, (b) IBN-4 extracted, (c) IBN-4-NH₂ and (d) IBN-4-HRP. Corresponding pore size distribution plots are shown in the inset figure. (B) Thermogravimetric analysis curves of (a) as-synthesized IBN-4; (b) IBN-4 extracted, (c) IBN-4-NH₂, (d) IBN-4-NH-GA and (e) IBN-4-HRP.

Figure 4

Fourier transform infrared spectroscopy (FT-IR) spectra of the (a) as-synthesized IBN-4 nanoparticles, (b) IBN-4 nanoparticles after surfactant removal (IBN-4-extracted), (c) IBN-4 nanoparticles modified with APTS groups (IBN-4-NH₂), (d) IBN-4-NH₂ nanoparticles modified with glutaraldehyde groups (IBN-4-NH-GA) and (e) IBN-4-NH-GA nanoparticles immobilized with HRP (IBN-4-HRP).

Figure 5

Ultraviolet–visible (UV-Vis) spectra and white-light sample images (inset) of (a) ninhydrin alone, (b) IBN-4 nanoparticles after surfactant removal, (c) IBN-4 nanoparticles modified with APTS groups (IBN-4-NH₂) and (d) IBN-4 nanoparticles modified with glutaraldehyde groups (IBN-4-NH-GA).

Figure 6

The viability of Human colon carcinoma (HT-29) cells: (A) treatment with IBN-4 loaded with Horseradish peroxidase (HRP) prepared via two synthetic routes; (B) treatment of IBN-4-HRP synthesized using two different routes in the presence or absence of indole-3-acetic acid (IAA) (IAA expressed in µM); and (C) treatment of various concentrations (200 and 500 μg/mL) of IBN-4-HRP synthesized in the presence or absence of IAA (IAA expressed in µM).

Figure 6

The viability of Human colon carcinoma (HT-29) cells: (A) treatment with IBN-4 loaded with Horseradish peroxidase (HRP) prepared via two synthetic routes; (B) treatment of IBN-4-HRP synthesized using two different routes in the presence or absence of indole-3-acetic acid (IAA) (IAA expressed in µM); and (C) treatment of various concentrations (200 and 500 μg/mL) of IBN-4-HRP synthesized in the presence or absence of IAA (IAA expressed in µM).

Figure 7

Bright field images of HT-29 cells without or with treatment of HRP-loaded IBN-4 nanoparticles and IAA: (a) Control (cells without treatment), (b) treatment with IAA (500 μM), (c) treatment with IAA (500 μM) and IBN-4-HRP (one-pot) (100 μg/mL) and (d) treatment with IAA (500 μM) and IBN-4-HRP (100 μg/mL).

Figure 8

Cytotoxic estimates of IBN-4 nanocomposites using an lactate dehydrogenase (LDH) leakage assay. Control experiment (CTL), P represents positive control (treated with Triton x-100) with various treatments using combinations of IAA and IBN-4-HRP.

Figure 9

Bright field images of HT-29 cells with or without treatment of IBN-4-HRP and IAA: (a) HT-29 cells without treatment; (b) treatment with IAA (1.6 mM); (c) treatment with IBN-4-HRP alone; and (d) treatment with IBN-4-HRP and IAA (1.6 mM).

Figure 10

Microphotographs of bright field and fluorescent microscopic views of HT-29 cells: (a) Bright field view of untreated control cells (undamaged cells); (b) fluorescent microscopic view of untreated control cells (undamaged cells); (c) bright field view of cells treated with IAA (1 mM) and IBN-4-HRP (100 μg/mL) (shrunken morphologies); and (d) fluorescent microscopic view of cells IAA (1 mM) and IBN-4-HRP (100 μg/mL) (damaged cells with elongated tails).